RNA_2014_比较了SILAC,iTRAQ和label-free鉴定剪切体蛋白
Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes
by metabolic labeling(SILAC),chemical labeling(iTRAQ), and label-free spectral count
CARLA SCHMIDT,1,4,7MADS GR?NBORG,1,5,7JOCHEN DECKERT,2,6SERGEY BESSONOV,2THOMAS CONRAD,2 REINHARD LüHRMANN,2and HENNING URLAUB1,3,8
1Bioanalytical Mass Spectrometry Group,2Department of Cellular Biochemistry,Max Planck Institute for Biophysical Chemistry,
37077G?ttingen,Germany
3Bioanalytics,Department of Clinical Chemistry,University Medical Center G?ttingen,37075G?ttingen,Germany
ABSTRACT
The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing.Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing.Here,we applied three independent mass spectrometry(MS)–based approaches for quantification of these proteins:(1)metabolic labeling by SILAC,(2)chemical labeling by iTRAQ,and(3)label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes.In total we were able to quantify157 proteins by at least two of the three approaches.Our quantification shows that only a very small subset of spliceosomal proteins (the U5and U2Sm proteins,a subset of U5snRNP-specific proteins,and the U2snRNP-specific proteins U2A′and U2B′′)remains unaltered upon transition from the B to the C complex.The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex.In terms of experimental procedure and the methodical aspect of this work,we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies.Moreover,we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique.
Keywords:spliceosome;quantitative proteomics;stable isotope labeling with amino acids in cell culture(SILAC);isobaric tags for relative and absolute quantification(iTRAQ);spectral count
INTRODUCTION
Proteomic analysis provides essential information about the function and regulation of protein complexes.Numerous mass spectrometry(MS)techniques have been developed to determine protein composition and their quantities (Aebersold and Mann2003;Steen and Mann2004;Ong and Mann2005;Domon and Aebersold2006;Yates et al. 2009;Bantscheff et al.2012;Nikolov et al.2012).To date, the relative quantification technique of stable-isotope label-ing followed by MS has frequently been used to quantify pro-teins from tissues,from different functional states of a cell, from compartments or from pull-down experiments involv-ing protein(–ligand)complexes.However,they have rarely been used to compare directly isolated native protein com-plexes that represent different functional states of a single molecular machine.Here,we examine the proteomes of iso-lated human precatalytic B and catalytically active C spliceo-somal complexes by quantitative MS.
The spliceosome catalyzes eukaryotic pre-mRNA splicing to generate mature mRNAs and is a highly dynamic and com-plex macromolecular machine.Spliceosomes consist of one or more of the five uridine-rich small nuclear ribonucleopro-tein particles(snRNPs;U1,U2,U5,the U4/U6di-snRNP,and the U4/U6.U5tri-snRNP),each of which contains U snRNA
4Present address:Department of Chemistry,Physical and Theoretical Chemistry Laboratory,University of Oxford,OX13QZ Oxford,UK
5Present address:Department of Beta Cell Regeneration,Hagedorn Research Institute,2820Gentofte,Denmark
6Present address:Axolabs GmbH,95326Kulmbach,Germany
7These authors contributed equally to this work.
8Corresponding author
E-mail henning.urlaub@mpibpc.mpg.de
Article published online ahead of print.Article and publication date are at https://www.wendangku.net/doc/5b8172415.html,/cgi/doi/10.1261/rna.041244.113.Freely available online through the RNA Open Access option.?2014Schmidt et al.This article,published in RNA,is available under a Creative Commons License(Attribution-NonCommercial3.0Unported), as described at https://www.wendangku.net/doc/5b8172415.html,/licenses/by-nc/3.0/.
METHOD
406RNA20:406–420;Published by Cold Spring Harbor Laboratory Press for the RNA Society
(s),a core set of seven Sm or LSm proteins,and numerous U snRNP-specific proteins.In vitro,several different functional states of the spliceosome can be distinguished;these are termed the E,A,B,B act,and C complexes(Wahl et al. 2009).Transitions between these different states are often ac-companied by dramatic rearrangements in the spliceosomal interactions.For instance,the precatalytic B complex,which contains the U4/U6.U5tri-snRNP,undergoes structural rear-rangements that destabilize the U1and U4snRNPs to form the activated B act complex;this then catalyzes the first step of splicing to generate the catalytically active C complex,which in turn catalyzes the second step of splicing to produce the ma-ture mRNA(Wahl et al.2009).Determining the protein com-position of each of the spliceosomal states has been a major goal in the past decade.Initial studies using active spliceo-somes assembled in vitro revealed that in addition to the U snRNP-specific proteins,more than100proteins are in-volved in the splicing cycle(Jurica et al.2002;Rappsilber et al.2002;Zhou et al.2002;Jurica and Moore2003).More de-tailed studies focused on analyzing the protein composition of the isolated functional states of the A,B,B act,and C complexes and the mRNPs in humans(Deckert et al.2006;Behzadnia et al.2007;Bessonov et al.2008,2010),yeast(B,B act,and C complexes)(Fabrizio et al.2009),and Drosophila(B and C complexes)(Herold et al.2009).MS analyses have revealed that the human B and C complexes contain approximately 130and150proteins,respectively.Of these,105proteins are stably associated with both complexes(Bessonov et al. 2008).The substantial change in composition from one func-tional spliceosomal state to another has been clearly shown by the determination of the protein compositions of isolated A,B,B act,and C complexes,each of which differs remarkably from the others even when isolated under native conditions that allow close comparison(Deckert et al.2006;Behzadnia et al.2007;Bessonov et al.2008,2010;Fabrizio et al.2009; Herold et al.2009).By using a novel2D gel electrophoresis sys-tem to quantify spliceosomal complexes,it has recently been shown that only60–70protein factors are moderately or high-ly abundant in the various spliceosomal complexes(Agafonov et al.2011).In these previous studies,the protein components of isolated spliceosomes were analyzed in a so-called“semi-quantitative”manner by determining the number of se-quenced peptides by MS(peptide count)or by measuring the intensity of stained proteins after2D gel electrophoresis to determine the relative and absolute abundances,respec-tively,of the proteins(Deckert et al.2006;Behzadnia et al. 2007;Merz et al.2007;Bessonov et al.2008,2010;Fabrizio et al.2009;Agafonov et al.2011).We applied now here—for the first time—three independent MS-based quantifica-tion methods(SILAC metabolic labeling[Ong et al.2002], iTRAQ chemical labeling[Ross et al.2004],and the label-free spectral count[Liu et al.2004])in determining the pro-tein quantities in purified spliceosomal B and C complexes. We identified and quantified approximately200proteins in both the B and the C complex preparations;this represents >95%of the previously published proteomes of the B and C complexes(Deckert et al.2006;Bessonov et al.2008). Strikingly,only a few of the proteins identified are“core”components of both complexes,the quantities of which do not change upon transition from B-to-C complex.These were a subset of U5snRNP proteins,the U2-specific proteins U2A′and U2B′′,and the evolutionarily conserved Sm pro-teins of U5and U2.Most proteins,in contrast,either joined or left the spliceosome during its transition from the precata-lytic B complex to the catalytically active C complex.This study expands our previous investigation of the proteomes of spliceosomal B and C complexes and may further be ex-tended to study the assembly kinetics of spliceosomal com-plexes in nuclear extract(NE).
RESULTS
For our quantitative MS studies,we used the same experimen-tal approach as Bessonov et al.(2008),namely,separation of the protein components of glycerol-gradient-purified spli-ceosomal complexes by1D gel electrophoresis,and thus re-duced the sample complexity to a similar extent(Bessonov et al.2008).Importantly,as a prerequisite of such an MS-based quantitative analysis,namely,comparing the exact amount of natively purified assemblies,we benefit from the measurement of the amount of32P-labeled pre-mRNA that is bound to the respective complexes.All quantitative MS-based analyses were performed with two biological replicates. We resumed the protein assignment previously introduced by Deckert et al.(2006),Bessonov et al.(2008),and Agafonov et al.(2011)and adjusted the protein classification of B and
C complexes according to our quantification results(Table
1).In addition,we list some proteins that could not unambig-uously be assigned to either of the two complexes,owing to their fluctuating quantification results(Supplemental Table 1).Finally,we identified and quantified several proteins that clearly show association with either the B or the C complex by all three approaches and were not classified as B-or C-spe-cific proteins previously by Bessonov et al.(2008)or Agafonov et al.(2011);Table1.
MS analysis of metabolically stable isotope-labeled (SILAC)spliceosomes
Metabolic labeling with stable isotopes(Ong et al.2002)is considered to be the gold standard in protein quantification; as proteins are fully labeled,samples can be pooled at an early stage of sample preparation and cleavage of proteins with endoproteinase trypsin always leads to coeluting labeled pep-tide pairs that are analyzed in the MS.We prepared spliceo-somes from HeLa NEs labeled with Lys+6,and Arg+10 (“heavy”NE)and spliceosomes prepared from NEs contain-ing the nonlabeled amino acids Lys+0and Arg+0(“light”NE)(Fig.1A).We had previously verified that the different SILAC NE preparations did not display significant differences Relative quantification of spliceosomal complexes
https://www.wendangku.net/doc/5b8172415.html,407
TABLE1.B:C protein ratios of spliceosomal proteins obtained by SILAC,iTRAQ,and spectral count
Protein MW
[kDa]Accession no.
B:C protein ratios
Complex
assignment SILAC#StDev iTRAQ#StDev
Spectral
count#StDev
U1snRNP?A
U1-A31.3gi|475915625.953/1 5.3422.35—/5 2.1415/7 4.83B U1-C17.4gi|450712727.15—/1OSB1/—B U1-70K51.6gi|295681037.515/6 6.11 5.1612/7 2.71 4.509/20.71B
17S U2snRNP?A,B,Bact
U2A′28.4gi|505930020.9224/250.210.9323/240.65 1.3660/440.83Core U2B′′25.4gi|45071230.9314/70.04 1.1414/120.86 1.1931/260.03Core SF3a12088.9gi|5032087 3.1511/320.00 2.6526/290.01 3.71104/280.45B SF3a6649.3gi|21361376 3.1713/20.19 6.027/6 2.938.0032/4 4.95B SF3a6058.5gi|5803167 3.2637/90.13 5.8024/14 1.25 4.5673/160.01B SF3b155145.8gi|54112117 3.9232/710.96 3.8268/1180.22 4.52244/540.29B SF3b145100.2gi|55749531 3.1225/420.24 4.3821/340.35 5.09117/230.14B SF3b130135.5gi|54112121 3.4031/940.00 4.2798/49 1.05 4.32397/92 1.28B SF3b4944.4gi|5032069 3.206/30.54 5.502/40.24 2.337/3 1.41B SF3b14a(p14)14.6gi|7706326 3.906/10 1.56 4.3111/140.08 2.3130/130.25B SF3b14b12.4gi|14249398 3.88—/4 2.8710/2 1.23 2.8334/2 1.73B
17S U2related
U2AF65?A,B53.5gi|6005926 1.511/— 3.632/—7.007/1B U2AF35?A27.9gi|5803207 2.861/— 5.598/—OSB2/—B hPRP43?A,B90.9gi|68509926 4.4251/250.38 4.2828/37 2.39 1.5992/580.26B SPF45?A,B45.0gi|1424967810.437/50.49 5.805/60.60OSB12/—B
SR140118.2gi|12293722712.3552/130.18 3.933/5 1.2611.0022/2 2.83B CHERP?A,B100.0gi|1192262607.3610/5 4.58 4.822/50.77OSB13/—B SF3b125103.0gi|4544674711.361/38.798.126/6 2.7322.0022/1B
U5snRNP
220K?B,Bact,C273.3gi|3661610 1.05107/1790.10 1.09154/1580.13 1.68661/
393
0.66Core
200K?B,Bact,C244.5gi|45861372 1.05128/2290.07 1.05211/1680.01 1.24581/
469
0.23Core
116K?B,Bact,C109.4gi|41152056 1.0092/650.11 1.0352/730.10 1.17299/
255
0.10Core
40K?B,Bact,C39.3gi|47585600.996/230.08 1.1820/160.22 1.5057/380.77Core 102K?B106.9gi|40807485 3.4018/530.11 2.2955/640.68 3.90226/58 1.15B 15K?B16.8gi|572980213.85—/2 5.883/3 1.6317.0017/1B 100K?B95.6gi|41327771 2.5828/230.29 1.3348/330.34 2.03128/630.03B 52K?B37.6gi|5174409 2.1710/80.21 2.283/5 1.72 3.7515/4 6.84B
U4/U6snRNP?B
90K77.6gi|475855617.8342/24 2.19 6.3237/39 2.758.13122/157.25B 60K58.4gi|4586137415.6416/4 2.027.0026/20 1.6610.1191/97.17B 20K20.0gi|54541548.675/8 2.12 2.533/40.04 3.0743/14 1.11B 61K55.4gi|4025486919.5913/3 3.23 6.4626/29 1.377.7077/108.88B
15.5K14.2gi|482686021.50—/213.042/2 5.5410.5021/2B
U4/U6.U5snRNP
110K?B90.2gi|139260689.0151/29 2.50 4.0523/190.63 6.50104/16 3.19B 65K?B65.4gi|56550051 3.2535/110.04 1.7021/190.11 1.8364/350.27B LSm proteins?B
LSm210.8gi|1086397720.881/7 3.58 4.864/80.5412.0024/2B LSm311.8gi|76573159.082/37.13 3.331/10.17 3.006/2B LSm415.4gi|691248612.983/3 4.77 5.158/20.53OSB19/—B LSm69.1gi|591915317.822/6 2.87 5.904/30.3628.0028/1B LSm711.6gi|770642319.622/— 5.158/2 2.068.008/1B LSm810.4gi|770642510.705/— 3.853/20.4113.0026/2B
Sm proteins?A,B,Bact,C
B24.6gi|4507125 1.605/190.14 1.7013/150.01 1.2953/410.05Core D113.3gi|5902102 1.8311/80.18 1.626/70.34 1.2436/290.44Core D213.5gi|29294624 1.7421/210.34 1.7535/220.18 1.0986/790.25Core D313.9gi|4759160 1.8117/90.12 1.7432/190.480.8550/590.14Core
(continued) Schmidt et al.
408RNA,Vol.20,No.3
TABLE1.Continued
Protein MW
[kDa]Accession no.
B:C protein ratios
Complex
assignment SILAC#StDev iTRAQ#StDev
Spectral
count#StDev
E10.8gi|4507129 1.888/70.21 1.6111/70.59 1.2239/320.91Core
F9.7gi|4507131 1.772/70.07 2.152/6 1.07 1.6922/130.16Core
G8.5gi|4507133 1.862/80.12 1.364/50.23 2.1317/8 1.37Core hPRP19/CDC5L complex
hPrp19?Bact,C55.2gi|76573810.3659/290.020.6061/620.040.59116/
197
0.21C
CDC5L?Bact,C92.2gi|110677470.4238/660.010.2145/300.030.44101/
229
0.04C
SPF27?Bact,C21.5gi|50316530.3428/170.010.5418/150.010.6728/420.50C
PRL1?Bact,C57.2gi|45058950.3326/110.010.8733/60.260.4739/830.13C
Hsp70?Bact,C70.4gi|57298770.1721/40.030.7215/100.670.2810/360.18C
AD-002?Bact,C26.6gi|77054750.278/90.080.186/30.050.448/180.35C CTNNBL1?Bact65.1gi|186447340.674/100.11 1.864/110.60 1.5021/140.41
hPRP19/CDC5L related
hSYF1?Bact,C100.0gi|557709060.2586/420.040.3362/650.080.4584/1880.15C CRNKL1?Bact,C100.6gi|307952200.2678/440.020.3988/820.110.43123/
286
0.05C
hIsy1?Bact,C33.0gi|201493040.20—/150.040.4016/170.280.188/450.01C SKIP?Bact,C51.1gi|69126760.3185/340.030.6058/420.030.5885/1470.24C RBM22?Bact,C46.9gi|89223280.2920/140.020.4935/390.060.3633/920.24C
Cyp-E?Bact,C33.4gi|51746370.2016/100.030.368/50.320.4716/340.08C
PPIL1?Bact,C18.2gi|77063390.318/210.090.4310/80.020.5821/360.10C KIAA0560?Bact,C171.3gi|387883720.25122/860.040.2496/1860.210.2573/2870.01C
G10?Bact,C17.0gi|32171175—/—0.6414/40.350.4221/500.12C hRES complex proteins
SNIP1?Bact45.8gi|213147200.73—/7 1.2310/20.61 1.3818/130.37Core MGC1213570.5gi|142493380.7510/140.01 1.4610/100.73 1.2436/290.28Core CGI-7939.7gi|49296270.628/60.15 1.063/20.42 1.148/70.14Core
B complex proteins
hPRP38?B37.5gi|24762236 2.70—/11 4.5011/70.64 6.8341/6B hSnu23?B28.8gi|133850468.852/4 4.77OSB10/—B
TFIP1196.8gi|8393259 1.5811/80.18 3.7512/22 3.88 1.0019/190.30B MFAP1?B51.9gi|50726968 2.6510/110.74 4.1538/110.87 5.0070/14 1.24B
RED?B65.6gi|10835234 5.5111/110.15 5.9223/20 1.8210.7586/80.09B hSmu-1?B57.5gi|8922679 6.129/8 4.78 4.8344/4 1.187.47127/1714.45B
RBM4250.3gi|2135995112.352/——/— 2.002/1B THRAP3?A108.6gi|482704010.215/119.39 1.6230/40.60 3.5868/190.17B
UBL58.5gi|13236510—/—13.561/2 4.92OSB12/—B HsKin1745.2gi|13124883 1.337/50.10 3.046/80.0821.0021/1B
Npw38BP70.0gi|7706501 3.532/20.608.046/17 1.0424.0024/1B
Npw3830.5gi|74735456 3.55—/17.68—/3OSB6/—B FUSE361.7gi|100816392 6.086/30.21 3.484/170.43 3.0010/1B
PUF6054.0gi|109087698 6.457/30.32 3.301/19 1.36OSB9/—B
RBM5/LUCA1556.12gi|6208720612.277/— 5.862/2 1.23OSB6/5B SAFB-like115.4gi|622440048.212/1 3.81 4.105/—12.0012/1B SFRS1259.4gi|2870379010.08—/1 4.601/6 2.55B
SPF3026.7gi|503211310.08—/530.161/—OSB5/—B
Step2factors
hPRP22?C139.3gi|48266900.1249/480.030.2155/110.110.0513/2570.01C hPRP18?C39.9gi|45061230.10—/10.391/40.01OSC—/5C hPRP17?Bact65.5gi|77066570.2628/290.040.3232/570.190.3431/910.12C hPRP16140.5gi|179995390.48—/20.43—/120.171/6C hSLU7?C68.4gi|274771110.326/140.110.1519/380.07OSC—/81C
C complex proteins
Abstrakt?C69.8gi|210710320.1625/240.050.1248/900.060.023/1640.01C GCIP p29?C28.7gi|463719980.0911/180.090.1811/70.000.042/45C
DDX35?C78.9gi|205441290.0613/150.010.2528/220.110.117/640.05C
(continued)
Relative quantification of spliceosomal complexes
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TABLE1.Continued
Protein MW
[kDa]Accession no.
B:C protein ratios
Complex
assignment SILAC#StDev iTRAQ#StDev
Spectral
count#StDev
Q9BRR8103.3gi|747329210.05—/70.293/90.150.041/23C c19orf29
(NY-REN-24)
88.6gi|1267231490.05—/210.1721/350.000.011/76C
PPIase-like3b18.6gi|195576360.022/70.010.248/30.10OSC—/21C PPWD1?C73.6gi|243080490.0818/180.000.1539/530.070.011/81C MORG134.3gi|1537912980.062/50.020.391/30.420.071/14C FRG129.2gi|47584040.223/20.000.58—/6OSC—/11C NOSIP?C33.2gi|77057160.10—/20.159/30.05OSC—/21C GPKOW?Bact,C52.1gi|158117820.215/120.170.666/—0.274/150.21C C1orf55?C39.3gi|1486642160.122/250.100.1019/140.03OSC—/41C FAM32A13.1gi|76616960.071/40.070.112/—0.091/11C Tip-4950.2gi|45067530.13—/——/—0.252/2C PPIG88.5gi|425602440.372/50.240.206/—0.122/17C FAM50A?C40.1gi|47582200.091/70.090.2215/50.100.031/36C FAM50B38.6gi|69123260.05—/20.252/30.16OSC—/19C C9orf78?C33.7gi|77065570.032/20.010.206/20.09OSC—/16C C10orf437.5gi|244320670.10—/50.102/60.010.091/11C CXorf56?C25.6gi|115458130.133/80.080.1612/110.110.021/51C DGCR1452.4gi|130276300.148/80.160.193/80.14OSC—/24C CCDC13044.7gi|135406140.10—/2—/—OSC—/7C NKAP47.0gi|133756760.061/50.050.226/—OSC—/14C ZCCHC1018.4gi|89231060.122/20.060.662/—OSC—/17C CDK1035.4gi|169506470.10—/60.223/40.03OSC—/9C TTC1488.2gi|334573300.211/80.190.278/110.07OSC—/19C NFKBIL143.1gi|267879910.08—/——/—0.201/5C NY-CO-10?Bact,C53.8gi|642764860.518/80.260.6612/20.030.6710/15 1.90C KIAA1604?Bact,C105.5gi|557497690.1917/260.040.4025/270.080.2728/1030.18C DDX34128.1gi|381580220.10—/50.377/200.12OSC—/24C NUFIP156.4gi|69125420.041/10.020.281/20.140.422/50.12C PRKRIP121.0gi|133759010.124/60.070.127/40.02OSC—/13C EJC/mRNP
eIF4A3?Bact,C46.9gi|76619200.2418/210.030.1531/—0.3233/1040.10C Magoh?C17.2gi|45050870.246/30.040.1810/50.040.205/250.08C Y14?C19.9gi|48269720.152/20.030.257/20.200.466/130.39C Pinin81.6gi|333561740.771/40.65 2.005/30.15 4.2517/40.35B UAP5649.1gi|18375623 3.185/4 4.10 3.2811/3 1.79 1.5323/150.82B
SR-related proteins
SRm160102.5gi|425423790.323/30.25 1.125/— 1.503/20.71
SRm300300.0gi|47590980.3313/260.100.977/40.930.1210/850.32
SR proteins
SF2/ASF?A,B,Bact,C27.8gi|5902076 3.5244/23 2.69 1.6314/8 1.170.9650/520.49
9G8?A,B,Bact,C27.4gi|72534660 2.9414/19 1.46 1.8724/9 1.00 1.51116/770.58
SRp2019.4gi|4506901 6.022/2 4.42 2.098/10.62 1.5235/230.77
SRp30c?Bact,C25.5gi|4506903 1.3216/180.020.7616/10.33 1.1742/360.53
SRp38?Bact,C31.3gi|57300790.956/— 1.1815/70.380.6741/610.04
SRp4031.3gi|3929378 1.3010/100.85 1.344/50.70 1.2535/280.25
SRp4631.2gi|15055543 4.15—/2 1.852/— 1.004/4
SRp5539.6gi|20127499 1.494/150.12 1.1117/70.33 1.1847/400.07
SRp7556.8gi|21361282 5.93—/2 1.16—/2
hTra-2alpha32.7gi|9558733 3.6115/6 1.01 2.129/20.97 1.6331/190.76
hTra-2beta?Bact,C33.7gi|4759098 4.752/16 1.10 1.3022/90.10 1.1045/410.24
hnRNP
hnRNP A1?A,B38.7gi|450444512.52—/120.56 4.326/2 1.2511.0022/28.49
hnRNP A3?A39.6gi|34740329 5.434/30.57 4.886/10.3717.0017/1
hnRNP A2/B137.4gi|14043072 6.101/8 4.49 4.116/40.737.0021/3
hnRNP C?C33.3gi|4758544 1.5141/280.24 1.8423/160.17 1.3577/570.74
hnRNP D38.4gi|1411042010.082/20.46—/— 4.004/1
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in their overall protein abundance,as shown by LC-MS/MS
analyses of peptides obtained from equivalent concentrations
of the“light”or“heavy”NE proteins hydrolyzed in gel
(Nikolov et al.2011).
In order to ensure that spliceosomal complexes isolated
from“light”and“heavy”NEs do not exhibit differences in
their activity,we monitored the splicing kinetics(Fig.2A)
and spliceosomal complex formation(Fig.2B)in“light”
and“heavy”SILAC NEs,as well as the RNA compositions
of the purified B and C complexes(Fig.2C).The analyses re-
vealed that catalytically active spliceosomes form in exactly
the same manner from“light”and“heavy”SILAC NEs,
with no significant quantitative differences in the overall pro-
tein composition or functional pre-mRNA splicing activities
(Fig.2).We then isolated spliceosomal B complexes from “light”SILAC NEs and C complexes from“heavy”SILAC NEs(Fig.1A),as described elsewhere(Bessonov et al.2008).
Proteins from the B and C complex mixtures were then sepa-
rated by1D SDS-PAGE and analyzed.We identified and
quantified266proteins(see Supplemental Fig.1);this includ-
ed all of the previously published spliceosomal proteins from
the B and C complexes,with the exception of the G10,UBL5,
RACK1,TOE1,RBM7,THOC3,and SC35proteins(Supple-
mental Table2).Additionally,we identified and quantified91
proteins that in previous studies had not been found to be
components of the spliceosome and that in most cases repre-
sent contaminating proteins commonly detected in large-
scale proteomic studies(Supplemental Table3).Chemical labeling of spliceosomal proteins
with iTRAQ reagents
We also applied chemical labeling with stable isotopes on pu-rified spliceosomal B and C complexes using iTRAQ reagents (Ross et al.2004).Chemical labeling is widely accepted as a quantitative MS-based method to analyze proteins derived from different samples.iTRAQ labeling of peptides from pu-rified spliceosomal B and C complexes was performed with a method most recently established after gel separation of pro-teins as described previously(Schmidt and Urlaub2009; Schmidt et al.2013).Extracted peptides from the B complex were labeled with iTRAQ-115and from the C complex with iTRAQ-116.Our iTRAQ analysis allowed us to quantify almost all previously published spliceosomal proteins from B and C complex preparations(Bessonov et al.2008),ex-cept for the proteins U1-C,RACK1,Tip-49,CCDC130, NFKBIL1,THOC3,DBPA,RBM42,and SC35(Supplemen-tal Table4).Similarly to the SILAC results,we found a total of 265proteins;among these were87proteins that had not been listed in the previous analysis by counting peptides;these mainly represented commonly encountered contaminants (Supplemental Table5).
Proteome analysis:spectral count
Previous analyses of protein components of spliceosomal complexes used the numbers of sequenced peptides in each
TABLE1.Continued
Protein MW
[kDa]Accession no.
B:C protein ratios
Complex
assignment SILAC#StDev iTRAQ#StDev
Spectral
count#StDev
hnRNP F45.7gi|148470406 3.85—/2 2.94—/10.753/4
hnRNP G47.4gi|56699409 3.5219/110.32 1.8732/60.49 1.2949/38 1.51
hnRNP H149.1gi|5031753 2.643/60.01 1.564/130.40 1.255/4
hnRNP K51.0gi|1416543512.0513/—1/20.74OSB24/—
hnRNP M77.5gi|14141152 3.568/1 1.04 3.498/40.04 3.0039/13 5.36
hnRNP Q69.6gi|15809590 2.493/— 1.34—/— 1.8917/90.24
hnRNP R70.9gi|5031755 1.0512/40.250.988/10.15 1.4423/160.88
hnRNP U?A90.6gi|1414116111.242/2 2.24 3.8610/— 2.008/40.71
PCBP1?A37.5gi|5453854 5.4211/20.14 4.845/100.26
PCBP238.1gi|14141166 4.4712/2 1.37 3.154/160.80 2.7452/19 1.54
RALY32.5gi|8051631 1.25—/8 1.644/3 1.190.5610/180.47
The average B:C protein ratio of two biological replicates after SILAC,iTRAQ,or spectral count quantification is shown.For SILAC and iTRAQ quantification,the number of peptide ratios(#)used for quantification is given for both biological replicates(1st replicate/2nd replicate).For spectral count,the sum of spectra from both biological replicates for B and C complexes is given(sum spectra B/sum spectra C).For all quan-tification methods,the standard deviation between the two biological replicates is provided.Note that if no spectra were acquired in one of the complexes,no standard deviation can be calculated for spectral count analysis.The proteins were classified as specific to complex B or to complex C,or as part of the spliceosomal core.Proteins that lacked an assignment could not be quantified by the respective approach. Proteins quantified by only one approach are not shown.For proteins identified solely in the B or C complex(OSB,only spectra B complex; OSC,only spectra C complex),no B:C protein ratio could be calculated by spectral count.The proteins are assigned according to the method of Bessonov et al.(2008)and have been regrouped on the basis of our quantification results.Proteins labeled with an asterisk are inferred to be major components of the human spliceosome and to be abundant in complexes A,B,C,or B act according to Agafonov et al.(2011). Proteins highlighted in gray were added to the list of spliceosomal B and C complex proteins according to results from this study.Protein ac-cession numbers were observed from NCBInr database.
Relative quantification of spliceosomal complexes
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sample to estimate the quantities of the proteins in their re-spective complexes (Deckert et al.2006;Behzadnia et al.2007;Bessonov et al.2008,2010;Fabrizio et al.2009;Herold et al.2009;Agafonov et al.2011).As in these studies spliceosomal complexes were purified to the highest stan-dards in biochemical terms (i.e.,kinetic splicing assays,density gradient centrifugation to obtain homogenous popu-lations),the number of peptides sequenced in the subsequent MS analysis was considered to be a valid approach for the es-timation of amounts of proteins in various complexes.Nonetheless,a certain ambiguity remained in these studies,namely,whether the peptide numbers and the respective comparison with numbers obtained from other complexes meet the requirements of quantitative protein analysis.As we have now obtained quantitative values for the proteins pre-sent in the spliceosomal B and C complex,we set out to com-pare these values with MS data obtained from a previous data set (Bessonov et al.2008).Instead of peptide count,we used the spectral count from the same data set of Bessonov et al.(2008)with the software Scaffold 2(Supplemental Table 5).The ratios of the spectra or peptides of B-complex proteins to C-complex proteins are listed in Supplemental Table 6(note that ratios could not be assigned to the proteins that were exclusively present in either the B or the C complex;these were labeled as “OSB ”[only spectra B complex]or “OSC ”[only spectra C complex]).This comparison revealed that assigning a protein abundance on the basis of peptide counting gives results consistent with those of spectral count-ing,with differences observed for only a few proteins (such as RACK1and Pinin)(Supplemental Table 6).Moreover,we ob-served,when compared with the values obtained from the la-beling experiments,good agreement in the quantification of the various proteins in the different complexes,so that it can be concluded —with a few exceptions that are discussed below —that spectral count (or even peptide count)is an ap-propriate method to quantify proteins in various spliceoso-mal complexes.
Validation of the quantification results
The investigated spliceosomal B and C complexes provide an ideal system for validation of the obtained quantitative re-sults.The expected theoretical B:C protein ratio for the cap-binding proteins (which interact with the 5′cap structure of the pre-mRNA)is 1:1;for the Sm proteins (which are core subunits for all of the U snRNPs except U6),2:1(since the C complex contains the U2and U5but lacks the U1and U4).Indeed,we observe a 1:1ratio for the CBP20and CBP80cap-binding proteins in the B:C complexes by all three methods (SILAC,iTRAQ,and label-free spectral count),with the exception of the spectral count for CPB20,which gave a ratio of 0.67(Supplemental Tables 2,4,6).For the B:C protein ratios for the Sm proteins,both iTRAQ and SILAC yielded an average protein ratio of 1.75,which is close to the expected value of 2(Fig.3A).However,spectral count gave the expected value only for the SmF and SmG proteins and showed clearly lower ratios for the other Sm proteins (Fig.3A).Overall,the quantification of these proteins pro-vided an internal validation of the different quantification methods.
A stable “core ”of U snRNP-specific proteins
The snRNAs of U2and the U5snRNPs remain stably associ-ated with the B and C complexes;however,only a subset of the U2-and U5-specific proteins appears in equal amounts within the two complexes (Fig.3B,C).SILAC,iTRAQ,and spectral count showed that the U2-A ′and U2-B ′′proteins had B:C ratios of approximately 1,whereas the U2snRNP-as-sociated splicing factors SF3a and SF3b were more abundant in the B complex (Table 1;Fig.3B).Likewise,only four of the eight U5snRNP proteins were found to be present in a 1:1ratio between the two complexes (220K,200K,116K,
and
FIGURE 1.Purification of the spliceosomal B and C complexes for proteomic analysis and iTRAQ and SILAC quantification.(A )B and C complexes were purified from “heavy ”or “light ”SILAC NEs,respec-tively.The complexes were allowed to assemble onto MS2-tagged pre-mRNA for 6or 180min,respectively.The complexes were then isolated by gradient centrifugation and affinity purification;isolated complexes were pooled in equal amounts;the proteins were separated by gel elec-trophoresis;and the peptides generated were analyzed by LC-MS/MS.(B )B and C complexes were purified from “normal ”(light)NE.The proteins were separated by gel electrophoresis,and after in-gel digestion,the peptides generated were analyzed by LC-MS/MS for proteomic anal-ysis,or the peptides generated from the B complex were labeled with iTRAQ reagent 115and those generated from the C complex were la-beled with iTRAQ reagent 116.After pooling,the samples were analyzed by LC-MS/MS (iTRAQ quantification).
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40K)(Fig.3C);we therefore consider the proteins U2A ′,
U2B ′′,U5-220K,200K,116K,and 40K together with the
Sm proteins of U2and U5snRNPs,which are also stably as-sociated with both complexes (see above,Fig.3A),to be the
spliceosomal “core ”proteins in the B and C complexes.
Specific proteins of the B and C complexes
Once we had validated our quantification results as described above,we next used the quantitative values of the protein abundances to investigate the correlation of their association with the B and C complexes.To do this,we consider proteins that show a B:C value above 2.0for at least two of the three methods to be specifically associated with the B com-plex and those showing a value below 0.5for at least two of the three methods to be specifically associated with the C complex.We also take the previous ordering and grouping of proteins into account (Bessonov et al.2008,2010;Agafo-nov et al.2011).Of note,to draw conclu-sion from the absolute number of the values,that means,whether,for example,an extremely high or low value (e.g.,U1-A,25.95[SILAC];PPIase-like 3b,0.02[SILAC])compared with a moderate val-ue (e.g.,hPrp19,0.36[SILAC];MFAP,2.65[SILAC])reflects the complete pres-ence or absence in B or C complex,re-spectively,cannot be unambiguously addressed.
Proteins predominantly associated with spliceosomal B complexes
During the transition from the B to the C complex,the U1and U4snRNPs with their associated proteins are destabilized and dissociate;this is clearly reflected by the high B:C ratios observed for these proteins (Table 1;Fig.4A,B).The U4/U6snRNP-specific proteins showed very high B:C ratios (average B:C 10.54),as de-termined by all three approaches (Fig.4B),showing that they,together with the U4snRNA,also dissociate from the
spliceosome during the transition from the B to the C complex.All three methods revealed high B:C ratios (>3)for the U6snRNP LSm proteins,showing that,al-though the U6snRNA remains associated with the C complex,the LSm proteins dis-sociate from U6during the B-to-C transi-tion (Table 1;Fig.4C).Contrary to the
U2A ′and U2-B ′′
proteins (see above),the U2snRNP-specific SF3a and SF3b
splicing factors were found to be more abundant in the B complex (Fig.3B)
and thus,however,do not seem to belong in the category of U snRNP “core ”proteins of the B and C spliceosomes.Likewise,U5snRNP-specific proteins 15K,52K,100K,and 102K show high B:C ratios as obtained by all three methods and thus represent B-specific proteins (Fig.3C).In addition,all proteins specific to the tri-snRNP (U4/U6.U5)showed high B:C ratios (Fig.4B;Table 1);this is consistent with pre-vious studies that have shown dissociation of some tri-snRNP –specific proteins from the spliceosome during its ac-tivation (Makarov et al.2002).For several non-snRNP proteins high B:C ratios were ob-served,showing that they are more abundant in B-complex preparations.Some examples are RED (average B:C 7.39),h-Smu-1(average B:C 6.12),and UBL5(B:C 13.56[iTRAQ]).By using our quantification methods,we classify these pro-teins as specific for the B complex (hPrp38,hSnu23,TFIP11,MFAP1,RED,hSmu-1,RBM42,TRAP3,UBL5,HsKin17,Npw38,Npw38BP)(Table 1;Fig.5A).In
addition
FIGURE 2.Metabolically labeled NEs retained full catalytic activity,as shown by analyzing the B and C complexes purified from “heavy ”or “light ”SILAC NEs,respectively.(A )The splicing ki-netics were determined from aliquots of splicing reactions taken from 0–180min and analyzed by denaturing gel electrophoresis.Pre-mRNA and splicing products were visualized by autoradiog-raphy.Splicing products first appeared after 10min.(B )The spliceosomal complex formation was assayed by native agarose gel electrophoresis and visualized by autoradiography.The A and B complexes were first observed after 2and 4min,respectively,while the C complex first appeared after 10–15min.(C )The RNA compositions of purified B (“light ”SILAC NE)and C (“heavy ”SILAC NEs)complexes were analyzed by denaturing gel electrophoresis and visualized by silver
staining (lanes 1,3)or autoradiography (lanes 2,4).B complexes contained U1,U2,U4,U5,and U6snRNA (lane 1)and a large amount of pre-mRNA (lane 2).C complexes contained U2,U5,and U6snRNA (lane 3)and splicing products and reduced amounts of pre-mRNA (lane 4).Relative quantification of spliceosomal complexes
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to these proteins,we thus extend the list of B-specific proteins by six proteins (FUSE3,PUF60,RBM5/LUCA15,SAFB-like,SFRS12,and SPF30)(Table 1),which were not present in the protein lists of previous studies (Bessonov et al.2008;Agafonov et al.2011).Proteins predominantly associated with C complexes
The so-called step 2splicing factors are required for the sec-ond step of pre-mRNA splicing,which occurs in the C com-plex (for review,see Umen and Guthrie 1995;Smith et al.2008),and accordingly,those proteins should be more abun-dant in this complex.Indeed,all of these proteins
(hPrp17,
FIGURE 3.Relative protein abundances of the Sm proteins and the U2and U5snRNP-specific proteins obtained by spectral count,SILAC,and iTRAQ.The B:C ratios are shown for the Sm proteins (A ),the U2snRNP-specific proteins (B ),and the U5snRNP specific proteins (C ).Ratios of proteins in B versus C complex are plotted on a logarithmic scale;error bars,SD between the two biological replicates.“1”indicates that a protein is present in these complexes in a 1:1ratio.The different shading of the bars represents the ratios of proteins in B and C complex-es derived from the values obtained by SILAC,iTRAQ,and spectral count,respectively (see Table 1;Supplemental
Tables).
FIGURE 4.Relative protein abundances in the B and C complexes for the U1,U4/U6,and U4/U6.U5snRNP-specific proteins and the LSm proteins,as obtained by SILAC,iTRAQ,and spectral count.(A )B:C ra-tios for U1snRNP-specific proteins.No protein ratios were obtained for U1-C from the spectral count since this protein was completely absent from the C complex.(B )B:C ratios for the U4/U6-and U4/U6.U5-spe-cific proteins.(C )B:C ratios for the LSm proteins.No protein ratio was obtained from the spectral count for LSm4since this protein was absent from the C complex.LSm5was not identified or quantified.For details,see legend to Figure 3.
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hPrp18,hPrp22,hPrp16,and hSlu7)had B:C ratios between 0.1and 0.5in iTRAQ and SILAC and close to 0by spectral count,showing that they were clearly associated with the cat-alytically active spliceosomal C complex (Fig.6A;Table 1).Additional proteins have previously been classified as “proteins recruited to the C complex ”and “potential C com-plex –specific proteins ”(Bessonov et al.2008).All of these proteins revealed low B:C ratios by all three quantification methods (Table 1;Fig.5B;Supplemental Tables 2,4,6).On the basis of these quantification results,we extended the list of C-specific proteins by three proteins (DDX34,NUFIP1,and PRKRIP1)(Table 1),all of which showed high abundance in the C complex,with B:C ratios between 0.04and 0.37.The DDX34protein,which was not previously identified within any spliceosomal complex,was clearly iden-tified as being more strongly represented in the C complex by both iTRAQ and SILAC.Spectral-count analysis identified this protein solely in the C complex but not in the B complex.
The hPrp19/CDC5L complex
The hPrp19/CDC5L (NTC in yeast,Chan et al.2003)complex is essential for pre-mRNA splicing and associates with the spliceosome before the first catalytic step of splicing (Ajuh
et al.2000).Together with U5snRNP,it forms the remodeled 35S U5complex in humans (Makarov et al.2002),and recent studies in yeast found it in the 35S ILS intron-lariat spliceosome (Fourmann et al.2013).Here,we found that all of these proteins (hPrp19,CDC5L,SPF27,PRL1,Hsp70,AD-002)—with the excep-tion of CTNNBL1,which showed an av-erage B:C ratio of 1.34(Table 1)—were clearly more abundant in the C complex than the B complex (Table 1;Fig.6B).The Npw38and Npw38BP proteins were previously found to comigrate at the top of the gradient during hPrp19/CDC5L complex purification,suggesting that they were co-isolated and/or not sta-bly associated with the hPrp19/CDC5L complex (Makarova et al.2004).These two proteins were found here to be highly abundant in the B complex compared with the C complex (Table 1;Fig.5A),demonstrating their predominant associ-ation with the B but not with C complex.
The RES complex
The RES (retention and splicing)com-plex consists of SNIP1,MGC12135,and CGI-79.It binds to the spliceosome be-fore the first step of splicing and is re-quired for efficient intron removal and
nuclear pre-mRNA retention (Dziembowski et al.2004).We found by all three quantification methods that the hRES proteins associate with both the B and C complexes,showing a 1:1B:C ratio (Table 1).Indeed,this is the only non-snRNP protein complex among the quantified non-snRNP splicing factors that remains constantly associated in the B and C complex.
The exon junction complex
The exon junction complex (EJC)protein complex binds spliced mRNAs in a sequence-independent manner close to site of exon –exon ligation.It is a highly dynamic complex with stably and more weakly associated protein components (Le Hir et al.2000;Lau et al.2003;Merz et al.2007;Singh et al.2012).We found that the EJC did not show the same B:C ratios for all its proteins.Only for three proteins (eIF4A3,Magoh,and Y14),we observed low B:C ratios (～0.25)(Table 1;Fig.6A),showing that they are much more abundant in the C complex,while proteins UAP56and Pinin are much more abundant in the B complex (B:C ratios of ～2.5)(Table 1).Other proteins previously assigned to the EJC showed fluctuating protein ratios (Supplemental Table 1),which did not allow us to classify them
unambiguously.
FIGURE 5.Relative protein abundances of the B-and C-specific proteins.(A )B:C ratios for B-specific proteins.(B )B:C ratios for C-specific proteins.For several proteins,no values for spectral count were obtained as these proteins had spectra only in the B (hSnu23,UBL5,Npw38,SPF30)or the C (PPIL3b,FRG1,NOSIP,C1orf55,FAM50B,C9orf78,DGCR14,CCDC130,NKAP,ZCCHC10,CDK10,TTC14,DDX34,PRKRIP1)complex.For details,see legend to Figure 3.
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DISCUSSION
In this study,we utilize three independent MS quantification techniques to compare the proteomes of stages of the spliceo-somal complex during its transition from a precatalytic to a catalytically active state.The various functional states of the spliceosome provide an ideal system to investigate quantita-tive MS approaches on native and functionally active molec-ular assemblies.First,the amount of complexes to be compared can be precisely controlled by measuring the amount of radioactively labeled pre-mRNA in each complex;second,the functionality of the complexes can be monitored by corresponding assays.
We found good overall agreement between results from the label-free spectral count and those from the two methods that use stable isotope labeling (SILAC and iTRAQ).The fact that our results are consistent among the different quantification approaches,in spite of the fact that we used different analytical conditions during the quantitative MS analysis (i.e.,different MS instruments and liquid chromatography conditions;see Materials and Methods)underlines the reproducibility of the methods employed here.Moreover,as previously generat-ed data sets (Bessonov et al.2008)were used to explore spectral counting as a label-free MS approach —and,importantly,gave results consistent with those of both the labeling approaches —this demonstrates that the evaluation of the protein abundance among various spliceosomal complexes as performed for A,B,B act ,and C in human spliceosomes and B,B act ,B ?,and C yeast spliceosomes is valid and indeed reflects the association of a distinct protein with its corresponding complexes (Deckert et al.2006;Behzadnia et al.2007;Bessonov et al.2008,2010;Fabrizio et al.2009;Agafonov et al.2011).
In agreement with previous comparative studies on MS-based quantification (Hendrickson et al.2006;Collier et al.2010;Li et al.2012),discrepancies between the isotope label-ing and the spectral count techniques were seen,especially for small proteins (<20kDa,e.g.,Sm proteins)(Table 1),for which accurate quantification was not achieved by spectral count owing to the limited number of peptides that were generated.The spectral count was also a little less accurate than labeling in quantifying proteins present in equal amounts within the B and C complexes,such as U5-220K (B:C 1.68),U5-40K (B:C 1.50),and CBP20(0.79)(see Sup-plemental Tables 2,4,6).Although the two methods based on stable isotope labeling yielded similar results,iTRAQ showed overall lower values for proteins strongly represented in the B complex and showed slightly higher values for pro-teins that are strongly represented in the C complex,than SILAC did.This may be due to the fact that precursor selec-tion for MS/MS is not 100%selective,allowing reporter ions of co-eluting peptides to contribute to the iTRAQ report-er ion intensity of the peptide being analyzed (Bantscheff et al.2007).
We compared our results with those from the previously published semi-quantitative analyses by Agafonov et al.(2011)and Bessonov et al.(2008)and found that all the stud-ies show the same results to a large extent.Remarkably,we found that most of the U snRNP-specific proteins are abun-dant in the B complex but not in the C complex.Here,we show that not only are U1and U4snRNP-specific proteins de-stabilized during the transition from complex B to complex C,but also U5and U2snRNP-specific proteins and LSm pro-teins.This leaves a U snRNP-specific protein “core ”in the B and the C complex consisting of only the Sm proteins,four U5snRNP-specific proteins (220K,200K,116K,and 40K),and two U2snRNP-specific proteins (U2A ′and U2B ′′).Evaluation of our quantitative data also allowed us to classify non-snRNP proteins as being specific for the B com-plex (Table 1).We extended the list of B-specific proteins by six proteins (FUSE3,PUF60,RBM5/LUCA15,SAFB-like,SFRS12,and SPF30)(Table 1),which showed high B:C ratios by at least two of the three methods.However,we have to take into account the fact that these proteins might be
residual
FIGURE 6.Relative protein abundances of the step 2factors,the EJC and the hPrp19/CDC5L complex –specific proteins obtained by SILAC,iTRAQ,and spectral count.(A )B:C ratios for step 2factors and the EJC.No protein ratio was obtained from the spectral count for hPrp18since this protein was absent from the B complex.(B )B:C ratios for the hPrp19/CDC5L complex –specific proteins.For CTNNBL1,no clear association to either the B or the C complex could be determined.For details,see legend to Figure 3.
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proteins belonging to the A complex,which was not included in our study.They might associate with the spliceosome at a very early point during pre-mRNA splicing and dissociate during formation of the catalytically active C complex.One example of this is the protein THRAP3,which is classified as B-specific by our results but was found to be specifically associated with the A complex by Agafonov et al.(2011). Along the same lines,we cannot make a clear statement re-garding the hnRNP proteins.Although these are more abun-dant in the B complex,we have no clear evidence as to whether they are B-specific or whether they are residual pro-teins from the H/A complex.
More precise conclusions can be drawn for C complex pro-teins.The hPrp19/CDC5L complex(Ajuh et al.2000; Makarova et al.2004)was found to be associated with the B complex but was shown to be more abundant in the C complex;we show here that the quantitative values are ap-proximately twice as great in the C as in the B complex,sug-gesting that this complex interacts loosely with the B complex and then becomes more stably associated with the C complex. Similarly,Agafonov et al.(2011)found the hPrp19/CDC5L complex to be less abundant in the B complex but highly abundant in the B act and C complexes.We did not analyze the intermediate complex B act in our study,and the slightly higher B:C ratio of the hPrp19/CDC5L proteins might reflect the association of these proteins during the B-to-C transition. In contrast,the second-step factors(hPrp16,hPrp17,hPrp18, hPrp22,and hSLU7)clearly show high abundance in the C complex with very low B:C ratios(～0.25),showing their asso-ciation with the activated C complex only.Only three EJC proteins(eIF4A3,Magoh,and Y14)were also found to be clearly associated with the C complex.Accordingly,we define other proteins showing very low B:C ratios as“C-specific pro-teins”(Table1).Some of these proteins have already been found to be abundant in the C complex by Agafonov et al. (2011);however,we are now able to extend the number of C-complex proteins(e.g.,c19orf29,FAM32A,c10orf4, DDX34,NUFIP1)(Table1).Importantly,DDX34has never been described in the context of spliceosomal complexes. This protein is a probable ATP-dependent RNA helicase(gi| 38158022),but its function has not yet been described,either in yeast or in humans.DDX34thus represents a potential tar-get for future studies.
Alongside the proteins that were clearly classifiable into B and C complex proteins,we identified some proteins that could not clearly be assigned owing to their inconsistent quan-tification values(Supplemental Table1).For these proteins, different B:C ratios were obtained by the different quan-tification approaches(e.g.,WDR70,SKIV2L2,DDX3) (Supplemental Table1).There are several possible reasons why these proteins do not show a clear quantitative associa-tion.First,they may represent transiently bound proteins that easily dissociate from the complexes during purification and are thus present to different extents in the different com-plex preparations.Examples are the pre-mRNA/mRNA-bind-ing proteins(Supplemental Table1),which showed very inconsistent quantitative trends and at least two of which (YB-1and ASR2B)were found to be present in all spliceoso-mal complexes(A,B,B act,and C)detected in each case at a dif-ferent abundance(Agafonov et al.2011).Second,they may be components of the B act complex,the intermediate complex in the B-to-C transition.We did not quantify the proteome of this complex,and the B:C ratios of proteins abundant in the B act complex fluctuate between different preparations and quantification approaches.Indeed,some of these proteins were found by Agafonov et al.(2011)to be specific for the B act complex,and a recent study in which the human B act com-plex was analyzed confirms this assumption,as several of these proteins were shown to be abundant in the B act complex only (Bessonov et al.2010);examples are hPrp2,PPIL2,RNF113A, MGC20398,and MGC23918(Supplemental Table1). CONCLUSIONS
We applied three independent MS-based quantification tech-niques to compare the proteomes of the precatalytic and the catalytically active spliceosomes(i.e.,B and C complexes). Overall,we confirm,but also extend,results from previous studies that addressed the relative abundances of proteins in the respective complexes by semi-quantitative approaches. We have found that the label-free spectral count technique provided a suitable method for quantifying highly purified samples(such as the spliceosome or other RNPs).However, it has its limits when quantifying proteins of low molecular weight or small fold changes.This is the first report of SILAC used to label and subsequently purify a molecular ma-chine that was functionally as active as its nonlabeled coun-terpart(as shown here for the spliceosome in terms of splicing efficiency and kinetics).Thus,it may be applicable to monitoring the assembly kinetics of the spliceosome in short time-frames in order to address the dynamic incorpo-ration and release of proteins in its various functional states or to gain insight into the changes of protein modification during the splicing cycle.Overall,iTRAQ(or a similar ap-proach using isotope-labeled reagents)is easier to apply to the quantitative investigation of spliceosomal proteins,in particular as a relatively large amount of NE is required for the assembly of spliceosomes.Thus,a reliable quantification technique based on chemical labeling with stable isotopes would be of benefit for in-depth quantitative analyses of spliceosomes from sources with low quantities. MATERIALS AND METHODS
Preparation of metabolically labeled“light”and “heavy”NEs
Dulbecco’s modified Eagle medium(DMEM;PAA Laboratories) lacking L-arginine and L-lysine was supplemented with10%(v/v) dialyzed fetal bovine serum(PAA Laboratories),1×penicillin/ Relative quantification of spliceosomal complexes
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streptomycin,and50mg/L of normal(“light”)or stable-isotope-la-beled(“heavy”)L-arginine and L-lysine.HeLa S3cells were grown in custom-made DMEM containing either“light”or“heavy”L-ar-ginine and L-lysine.Cells were grown for at least six passages at100 mL(～0.5×108to1.0×108cells/mL)in200mL spinner flasks.The cells were then expanded to higher volumes and transferred to a2.5 L fermenter.The cells were grown under standard conditions(50 mg/L L-arginine and L-lysine)with continuous perfusion of medi-um(0.5–1.0v/24h).A total of1.5L(5×106cells/mL)of cells was harvested,and NEs were prepared according to the method of Dignam et al.(1983)from“light”-or“heavy”-labeled cells.L-argi-nine and L-lysine,and stable isotope-labeled L-arginine(13C615N4; Arg+10)and L-lysine(13C6;Lys+6),were purchased from Sigma-Aldrich.
Generation of aptamer-tagged pre-mRNA
and in vitro splicing
MS2-tagged PM5pre-mRNA was generated as previously described (Deckert et al.2006;Bessonov et al.2008).In vitro splicing was per-formed in either“light”or“heavy”HeLa NE,using32P-labeled, m7G(5′)ppp(5′)G-capped,and MS2-tagged PM5pre-mRNA. RNA was separated on a8.0M urea–10%(v/v)polyacrylamide gel.Spliceosomal complex assembly was analyzed by native gel elec-trophoresis on a2%(m/v)agarose gel.
Affinity selection of spliceosomal B and C complexes Spliceosomal B and C complexes for proteomic analysis and SILAC and iTRAQ quantification were isolated as previously described (Bessonov et al.2008).Briefly,32P-labeled,m7G(5′)ppp(5′)G-capped,and MS2-tagged PM5pre-mRNA was incubated with MS2-MBP fusion protein.Spliceosomal complexes were allowed to assemble from“light”or“heavy”NEs for6min(B complex) or180min(C complex).For SILAC quantification,B complexes were assembled from“heavy”NEs,and C complexes were assem-bled from“light”NEs.Assembled complexes were separated on 10%–30%(v/v)glycerol gradients,and40–45S gradient fractions were subjected to affinity selection on amylose beads.
Sample preparation and MS for SILAC quantification Three pmoles of affinity-purified B and C complexes was mixed in equal amounts according to the32P-labeled pre-mRNA.Proteins were separated by gel electrophoresis on a4%–12%Bis-Tris precast gel(NuPAGE,Invitrogen)and stained with colloidal Coomassie blue.The entire gel lane was cut into25pieces,and proteins were digested in-gel as described previously(Shevchenko et al.1996). Samples were redissolved in10%(v/v)acetonitrile/0.15%(v/v)for-mic acid(FA)and analyzed on a CAP-LC system coupled to a Q-ToF Ultima mass spectrometer(Waters)or on an Agilent HP 1100series system coupled to a LTQ-Orbitrap XL(Thermo Fisher Scientific).
Sample preparation and MS for iTRAQ or spectral count quantification
Four pmoles each of affinity-purified B and C complexes were sep-arated by8%/14%(v/v)SDS-PAGE,respectively,and stained with Coomassie blue.Entire gel lanes were cut into60–70slices. Proteins were digested in-gel as described previously(Shevchenko et al.1996)except that50mM triethylammonium bicarbonate buff-er(TEAB,Sigma-Aldrich)instead of ammonium bicarbonate buffer was used for the iTRAQ preparation.For iTRAQ labeling,the ex-tracted peptides were dissolved in20μL100mM TEAB buffer. Internal standards were prepared by mixing5μL TEAB buffer with5μL aliquots of samples generated from gel slices cut at the same height from both gel lanes.iTRAQ reagents were reconstituted at room temperature in70μL ethanol per vial.iTRAQ reagent(5μL) was added to each sample,and samples were incubated at room temperature for1h with gentle mixing.Internal standards were la-beled with iTRAQ-114,and samples from the B and C complexes were labeled with iTRAQ-115and iTRAQ-116,respectively.The re-maining iTRAQ reagent was quenched by adding5μL of50mM glycine and incubating at room temperature for30min with gentle mixing.Samples to be compared,such as those containing the pep-tides generated from B and C complexes,were pooled with their rel-evant internal standards and then dried in a vacuum centrifuge (Schmidt and Urlaub2009).For the MS analysis,iTRAQ or spectral count samples were dissolved in10%(v/v)acetonitrile with0.15% (v/v)FA and subsequently analyzed on a Waters Q-TOF Ultima coupled to a Waters CAP-LC system.
LC-coupled ESI MS–MS/MS on a Q-ToF mass spectrometer
To analyze samples over the CAP-LC system coupled to the Q-ToF Ultima mass spectrometer(Waters),peptides were separated online by reversed-phase chromatography using0.1%(v/v)FA as mobile phase A and80%(v/v)acetonitrile/0.15%(v/v)FA as mobile phase B.The peptides were loaded onto a trap column(μ-Precolumn Cartridge,Acclaim PepMap100C18,300μm i.d.×5mm,LC Packings)and separated at a flow rate of200nL/min on an analytical column packed in-house(C18,Reprosil,Maisch)with a gradient of 7%–40%mobile phase B over50min.Eluted peptides were ana-lyzed directly in the Q-ToF mass spectrometer in a data-dependent manner.MS scans were acquired for1sec followed by three MS/MS spectra for3sec,each with an ion-mass window set to2.5Da.The MS-to-MS/MS switch was set to15counts/sec,and the MS/MS-to-MS was set to an intensity below a threshold of2counts/sec.Charge state recognition was used to estimate the collision energy for the se-lected precursors.Scan time and interscan time were set to0.9sec and0.1sec,respectively.Peak lists were generated from raw data by using MassLynx v4.0software with the following settings:smooth window4.00,number of smooth2,smooth mode Savitzky-Golay, percentage of peak height to calculate centroid spectra at80% with no baseline subtraction.
LC-coupled ESI MS–MS/MS on a LTQ-Orbitrap
mass spectrometer
Samples were analyzed on an HP1100series system(Agilent)cou-pled to a hybrid Linear Ion Trap–Orbitrap mass spectrometer (LTQ-Orbitrap XL,Thermo Fisher Scientific).Peptides were sepa-rated by online reversed-phase nanoflow chromatography,using 0.1%(v/v)FA as the mobile phase A and95%(v/v)acetonitrile/ 0.1%(v/v)FA as the mobile phase B.Peptides were loaded onto a trap column packed in-house(1.5cm,360μm o.d.,150μm i.d.,
Schmidt et al.
418RNA,Vol.20,No.3
ReproSil-Pur C18-AQ,5μm,Dr.Maisch)and separated at a flow rate of300nL/min on an analytical C18capillary column(30cm, 360μm o.d.,75μm i.d.,ReproSil-Pur C18-AQ,5μm)with a gradi-ent of0%–38%mobile phase B over35min.Eluted peptides were analyzed directly in the mass spectrometer(LTQ-Orbitrap XL; Thermo Fisher Scientific).The LTQ-Orbitrap was operated in a data-dependent mode.Survey full-scan MS spectra were acquired in the LTQ-Orbitrap(m/z350–1400)with a resolution of30000 at m/z400,and an automatic gain control target of5×105.The five most intense ions were selected for CID(collision-induced dis-sociation)MS/MS fragmentation and detection in the linear ion trap,with previously selected ions dynamically excluded for60 sec.Singly charged ions and ions with unrecognized charge states were also excluded.Internal calibration of the Orbitrap was per-formed using the lock mass option(lock mass:m/z445.120025) (Olsen et al.2005).Mascot generic format(mgf)files were generat-ed from raw data using Mascot Daemon v2.2.2(Matrix Science). Data analysis and quantification
Peak lists generated were searched against NCBI nonredundant da-tabase(October8,2007;5539442sequences),by using Mascot v.2.2.04as search engine.The mass accuracy filter used was0.2 Da for the parent and fragment ions for the Q-ToF mass spectrom-eter.For the Orbitrap mass spectrometer,this was5ppm for precur-sor and0.5Da for product ions.Peptides with no or at most two missed cleavage sites were defined as tryptic peptides.Carbamido-methylation of cysteines and oxidation of methionine residues were allowed as variable modifications.For SILAC and iTRAQ quantification,“heavy”arginine(Arg+10)and“heavy”lysine(Lys+6) and iTRAQ modifications,respectively,were allowed as fixed modifications.
SILAC quantification
SILAC quantification was carried out by using unique peptides with the MSQuant software v1.2.Data normalization was performed on proteins known to be present in a1:1ratio(e.g.,the5′pre-mRNA cap-binding proteins CBP20and CBP80,the U5-220K and U5-200K proteins).
iTRAQ quantification
Non-normalized peptide ratios for iTRAQ quantification were ob-tained from Mascot v2.2.04for unique peptides with a minimum peptide score of20.Proteins were quantified from the main bands by calculating the mean ratio after manual removal of outliers. Data normalization was performed on proteins known to be present in a1:1ratio,as above.Protein ratios obtained were further validated by three independent procedures:(1)calculating the labeling effi-ciency for each protein in each band;(2)using the same amounts of nonmodified trypsin(Roche),resulting in a1:1ratio for autopro-teolytic trypsin peptides;and(3)analyzing peak intensities of the reporter ions for the internal standards(iTRAQ-114)of low-scor-ing peptides.As the internal standard was prepared by pooling aliquots from iTRAQ-115–and iTRAQ-116–labeled samples the following equation represents the intensity ratios:intensity [iTRAQ-114]=?intensity[iTRAQ-115]+?intensity[iTRAQ-116]whereby[iTRAQ?114]=(5μL/(20μL?5μL))[iTRAQ ?115]+(5μL/(20μL?5μL))[iTRAQ-116](see also Schmidt and Urlaub2009).
Spectral count
Unweighted spectral count for proteins identified in B and C com-plexes was obtained by using the software Scaffold2.B:C protein ra-tios were calculated manually from the obtained number of spectra for each protein.
SUPPLEMENTAL MATERIAL
Supplemental material is available for this article.
ACKNOWLEDGMENTS
We thank P.Kemkes,H.Kohansal,M.Raabe,and U.Plessmann for excellent technical assistance;I.Lemm for help with SILAC cell cul-ture;and V.A.Raker for help in manuscript preparation.M.G.was supported by the Danish Agency for Science,Technology and Innovation(DASTI).This work was supported by grants from the Deutsche Forschungsgemeinschaft(SFB860)to R.L.and H.U. Received July11,2013;accepted December10,2013.
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第10 章模拟比较器和ADC 接口 Program type : Application Clock frequency : 4.000000 MHz Memory model : Small External SRAM size : 0 Data Stack size : 256 *****************************************************/ #include
鉴相器原理与分类
鉴相器原理及分类更新于2010-05-13 03:52:41 文章出处:与非网 鉴相器取样鉴频 鉴相器-原理特性 使输出电压与两个输入信号之间的相位差有确定关系的电路。表示其间关系的函数称为鉴相特性。鉴相器是锁相环的基本部件之一，也用于调频和调相信号的解调。常见的鉴相特性有余弦型、锯齿型与三角型等。 鉴相器特性用ud(t)＝kdf【θe(t)】表示。式中kd为鉴相器的增益系数；θe(t)＝θ1(t)－θ2(t)，表示两个输入信号之间的相位差。函数f【·】表示鉴相特性，它反映鉴相器的输出电压ud(t)与相位差的关系。常见的鉴相特性有余弦型、锯齿型与三角型等。 鉴相器-分类 鉴相器可以分为模拟鉴相器和数字鉴相器两种。 二极管平衡鉴相器是一种模拟鉴相器。两个输入的正弦信号的和与差分别加于检波二极管，检波后的电位差即为鉴相器的输出电压。其鉴相特性通常为余弦型的。鉴频鉴相器是一种数字鉴相器。两个输入信号是脉冲序列，其前沿（或后沿）分别代表各自的相位。比较这两个脉冲序列的频率和相位即可得到与相位差有关的输出。这种鉴相器的鉴相特性为锯齿形。因它兼具鉴频作用，故称鉴频鉴相器 二极管平衡鉴相器 这是一种模拟鉴相器，原理电路如图1。二极管D1、D2和C1R1、C2R2构成两个峰值检波器。两个输入的正弦信号u1(t)=U1sin(ωt+θ1)、u2(t)＝U2sin(ωt+θ2)的和与差分别加于检波二极管D1和D2，检波后的电压差即为鉴相器的输出电压ud。当U2U1时,ud∝U1cos(θ1－θ2)。在这种情况下,它的鉴相特性是余弦型的(图2a)。 鉴频鉴相器 这是一种数字鉴相器。两个输入信号是脉冲序列，其前沿(或后沿)分别代表各自的相位。比较这两个脉冲序列的频率和相位即可得到与相位差有关的输出。图3是一种鉴频鉴相器的框图。比相器可由触发器构成。当两个输入信号u1和u2同频同相时,触发器没有输出,充电电流等于零。当u1脉冲序列超前于u2时，触发器产生一个其宽度与相位差成正比的正脉冲，充电电路被充电，其输出电压为正值，大小与充电脉冲宽度成正比。若u1落后于u2,则触发器输出一个负脉冲，充电电路的输出为负值。这种鉴相器的鉴相特性为锯齿形（图2b）。这种鉴相器兼具鉴频作用，故称鉴频鉴相器。
数值比较器的应用
数值比较器电路的仿真分析及应用 程勇 陈素 陈淑平 （机电信息工程系 实训中心 450008） 摘要：数值比较器是数字电路中经常用到的典型电路，传统的教学模式中，对数值比较器的学习及应用设计，离不开在实验室中的电路调试，学习方式较为枯燥抽象，又耗时费力，学习效果也不尽理想。现代电子设计中，由于仿真软件的出现，变抽象的知识为直观的展示，既可以通过仿真学习数值比较器的工作原理，又可以通过仿真进行数值比较器的应用设计，学习及应用效果事半功倍。 关键词：数值比较器、仿真分析、应用 在各种数字系统尤其是在数字电子计算机中，经常需要对两个二进制数进行大小判别，然后根据判别结果转向执行某种操作。用来完成两个二进制数的大小比较的逻辑电路称为数值比较器，简称比较器。在数字电路中，数值比较器的输入是要进行比较的两个二进制数，输出是比较的结果。 一．电路设计分析 首先讨论1位数值比较器。1位数值比较器是多位比较器的基础。当A 和B 都是1位二进制数时，它们的取值和比较结果可由1位数值比较器的真值表表示，如表1所示。 表1 1位数值比较器的真值表 由真值表可得如下逻辑表达式 A B A B A B F AB F AB F AB AB A B ><====+=⊕ 由逻辑表达式可以画出如图1所示的逻辑图。
图1 1位数值比较器逻辑图 二．比较器电路的仿真分析 （一）元件选取及电路组成 打开仿真软件Multisim 10，根据图1所示的1位数值比较器逻辑图，可以在仿真软件Multisim 10中构建仿真电路，如 图3所示。 1．元件选取 （1）指示灯的选取 1位数值比较器逻辑运算完后，输出结果处 接一指示灯作为指示，灯亮表示运算结果成立， 灯灭表示运算结果不成立。单击元件栏的Place Indicator→PROBE，选取PROBE_RED指示灯。 为了观察清晰明白，将指示灯PROBE连击打开其图2 指示灯的Label设置 设置对话框，在其Label中的标号由默认的X1改为“A等于B”、“A大于B”、“A 小于B”等。如图2所示。 （2）其他元器件可参照以下说明取用。 电源VCC：Place Source→POWER_SOURCES→VCC 接地：Place Source→POWER_SOURCES→GROUND，选取电路中的接地。 或非门U1A的选取：Place TTL→74LS→74LS02D 与门U3A、U5A的选取：Place TTL→74LS→74LS08D 非门U2 A、U4A的选取：Place TTL→74LS→74LS04N 2．电路组成 参照图3放置元件并进行连接，构成1位数值比较器的仿真测试电路。 （二）仿真分析
任务4-6 鉴频与鉴相
任务4-7 鉴频与鉴相 4-6-1资讯准备 任务描述 1．了解鉴频的概念、方法及鉴频的主要技术要求； 2．理解各类鉴频电路的组成、工作原理、分析方法及主要特点。 资讯指南 导学材料 一、鉴频方法综述 调频信号解调又称为频率检波，是从调频波中取出原调制信号，即输出电压与输入信号的瞬时频率偏移成正比，又称为鉴频器，简称鉴频。它是把调频信号的频率 )()(t t c ωωω?+=与载波频率c ω比较，得到频差)()(t f t m ωω?=?，从而实现频率检波。 1．鉴频的方法 鉴频的方法很多，其工作原理都是将输入的调频信号进行特定的变换，使变换后的波形包含反映瞬时频率变化的量，再通过低通滤波器滤波就可以得到原调制信号。常用的鉴频方法有以下几种： (1)斜率鉴频器 它先将输入等幅的调频波通过线性网络进行频率-幅度变换，得到振幅随瞬时频率变化的调频波，然后用包络检波器将信号的振幅变化取出来；其输出信号就是原调制信号。 (2)相位鉴频器 它先将输入等幅的调频波通过线性网络进行频率-相位变换，得到附加相位随瞬时频率变化的调频波，然后用鉴相器将它的附加相移变化取出来，其输出信号就是原调制信号。
(3)脉冲计数式鉴频器 它先将输入等幅的调频波通过非线性变换网络进行波形变换，得到数目与瞬时频率成正比、但幅度和形状相同的调频脉冲序列，然后将信号通过低通滤波器，其输出信号就是原调制信号。 2．鉴频器的主要技术要求 鉴频器的输出电压u o随输入调频的瞬时频率f的变化特性称为鉴频特性。为了实现不失真的解调，u o应与f成线性关系，即鉴频特性曲线应为一条直线。但是，实际的鉴频特性往往是一条曲线，所以它只能在有限频率范围内实现线性鉴频。图4-6-1为一典型的鉴频特性曲线，由于该曲线与英文字母“S”相似，故又称为S曲线。由图可以看出，对应于调频波的中心频率f c，输出电压u o=0；当信号频率向左右偏离时，u o分别为正负值。 图4-6-1 鉴频特性曲线 对鉴频器主要技术要求有： (1)鉴频特性为线性 鉴频电路输出低频解调电压与输入调频信号瞬时频偏的关系称为鉴频特性，理想的鉴频特性应是线性的。实际电路的非线性失真应该尽量减小。 (2)鉴频线性范围要宽 由于输入调频信号的瞬时频率是在载频附近变化，故鉴频特性曲线位于载频附近，其中线性部分称为鉴频线性范围。要求其鉴频线性范围足够宽。 (3)鉴频灵敏度要高 在鉴频线性范围内，单位频偏产生的解调信号电压的大小称为鉴频灵敏度S d。S d越大，鉴频效率就越高。 二、鉴频电路 1．斜率鉴频器 斜率鉴频器是利用频幅转换网络将调频信号转换成调频-调幅信号，然后再经过检波电路取出原调制信号，这种方法称为斜率鉴频，因为在线性解调范围内，解调信号电压与调频信号瞬时频率之间的比值和频幅转换网络特性曲线的斜率成正比。斜率鉴频的电路模型如图4-6-2所示。
乘积型相位鉴频器的设计
一、电路原理 1．电路原理 （1）乘积型相位鉴频由移相网络、乘法器和低通滤波器三部分组成。调频信号一路直接加至乘法器，另一路经相移网络移相后(参考信号)加至乘法器。由于调频信号和参考信号同频正交，因此，称之为正交鉴频器。如图所示。 图1 正交鉴频原理图 （2）用LM1596构成的乘积型相位鉴频器电路如图所示。 图2 LM1596构成的相位鉴频器 其中C 1与并联谐振回路C 2L 共同组成线性移相网络，将调频波的瞬时频率的变化转变成瞬时相位的变化。分析表明，该网络的传输函数的相频特性)(ωφ的表 达式为： )]1(arctan[2)(20 2 --=w w Q w π φ 当 <
或 )2arctan(2 )(0 f f Q f ?-= ?π φ 式中f 0—回路的谐振频率，与调频的中心频率相等。Q —回路品质因数。△ f —瞬时频率偏移。相移φ与频偏△f 的特性曲线如图所示。 图3 相移φ与频偏△f 的特性曲线 2．主要技术指标 相位鉴频法的原理框图如下图所示。图中的变换电路具有线性的频率—相位转换特性，它可以将等幅的调频信号变成相位也随瞬时频率变化的、既调频又调相的FM-PM 波。把此FM-PM 波和原来输入的调频信号一起加到鉴相器上，就可以通过鉴相器解调此调频信号。相位鉴频法的关键是相位检波器，相位检波器或鉴相器就是用来检出两个信号之间的相位差，完成相位差—电压变换作用的部件或电路。设输入鉴相器的两个信号分别为： 把它们同时加于鉴相器，鉴相器的输出电压o u 是瞬时相位差的函数，即： 在线性鉴相时，o u 与输入位相差21()()()e t t t ???=-成正比。信号2u 中引入/2π固 定相移的目的在于当输入相位差21()()()e t t t ???=-在零附近正负变化时，鉴相器输出电压也相应地在零附近正负变化。 图4 相位鉴频器的框图 11122222cos ()cos ()sin ()2c c c u U t t u U t t U t t ω?πω?ω?=+???? ?? =-+=+???????? 21()()o u f t t ??=-????
比较器
东南大学电工电子实验中心 实验报告 课程名称：电子电路实践 第六次实验 实验名称：比较器电路 院（系）：自动化专业：自动化姓名：学号： 实验室: 103 实验组别： 同组人员：实验时间：2012 年 5 月3日评定成绩：审阅教师：
实验六比较器电路 一、实验目的 1、熟悉常用的单门限比较器、迟滞比较器、窗口比较器的基本工作原理、电路特性和主要 使用场合； 2、掌握利用运算放大器构成单门限比较器、迟滞比较器和窗口比较器电路各元件参数的计 算方法，研究参考电压和正反馈对电压比较器的传输特性的影响； 3、了解集成电压比较器LM311的使用方法，及其与由运放构成的比较器的差别； 4、进一步熟悉传输特性曲线的测量方法和技巧。 二、实验原理 2、窗口比较器电路如上图所示，它由同相比较器A1、反相比较器A2及二极管D1，D2组成。该电路的功能是，可以判别输入电压V i是否介于下参考电压V RL与上参考电压V RH之间(所谓的窗口)。如果V RL＜V i
3、集成电压比较器比集成运放的开环增益低，失调电压大，共模抑制比小；但其响应速度快，传输延迟时间短，而且不需外加限幅电路就可直接驱动TTL、CMOS和ECL等集成数字电路；集成电压比较器的输出方式分为普通、集电极（或漏极）开路输出或互补输出三种情况。LM311为集电极（或漏极）开路输出，必须在输出端接一个电阻至电源才能正常工作。下图为LM311作为单门限电压比较器的典型电路，其中R PU为上拉电阻。 三、预习思考 1、用运算放大器LM741设计一个单门限比较器，将正弦波变换成方波，运放采用双电源 供电，电源电压为±12V，要求方波前后沿的上升、下降时间不大于半个周期的1/10，请根据LM741数据手册提供的参数，计算输入正弦波的最高频率可为多少。 答：查询LM74的数据手册，可得转换速率为0.5V/us，电源电压为±10V左右，计算可得输出方波的最大上升时间为40us，根据设计要求，方波前后沿的上升下降时间不大于半个周期的1/10，计算得信号的最大周期800us，即最高频率1.25KHz。 2、画出迟滞比较器的输入输出波形示意图，并在图上解释怎样才能在示波器上正确读出上 限阈值电平和下限阈值电平。 答: Ch1接输入信号，ch2接输出信号，两通道接地，分别调整将两个通道的零基准线，使其重合。用示波器游标功能，通道选择CH1，功能选择电压，测出交点位置处电压即对应上限和下限阈值电平。 3、查阅LM311的数据手册，列表记录其主要参数，并做简单解释。
电压比较器电路图
电压比较器电路。 电压比较器是比较两个电压和开关输出或高或低的状态，取决于电压较高的电路。一个基于运放电压比较器上显示。图1显示了一个电压比较器的反相模式图显示了在非反相模式下的电压比较。 电压比较器 非反相比较 在非反相比较器的参考电压施加到反相输入电压进行比较适用于非反相输入。每当进行比较的电压(Vin)以上的参考电压进入运放的输出摆幅积极饱和度(V+)，和副反之亦然。实际上发生了什么是VIN和Vref(VIN-VREF)之间的差异，将是一个积极的价值和由运放放大到无穷大。由于没有反馈电阻Rf，运放是在开环模式，所以电压增益(AV)将接近无穷。+所以最大的可能值，即输出电压摆幅，V。请记住公式AV=1+(Rf/R1)。当VIN低于VREF，反向发生。 反相比较
在相比较的情况下，参考电压施加到非反相输入和电压进行比较适用于反相输入。每当输入电压(Vin)高于VREF，运放的输出摆幅负饱和。倒在这里，两个电压(VIN-VREF)之间的差异和由运放放大到无穷大。记住公式AV=-Rf/R1。在反相模式下的电压增益的计算公式是AV=-Rf/R1.Since没有反馈电阻，增益将接近无穷，输出电压将尽可能即负，V-。 实际电压比较器电路 一种实用的非基于UA741运放的反相比较器如下所示。这里使用R1和R2组成的分压器网络设置参考电压。该方程是VREF=(五+/(R1+R2)的)×R2的。代入这个方程电路图值，VREF=6V。当VIN高于6V，输出摆幅?+12V直流，反之亦然。从A+/-12V 直流双电源供电电路。 电压比较器的使用741
一些其他的运放，你可能会感兴趣的相关电路 1求和放大器：总结放大器可以用来找到一个信号给定数量的代数和。 2。集成使用运放：对于一个集成的电路，输出信号将输入信号的积分。例如，一个集成的正弦波使余弦波，方波一体化为三角波等。 3。反相放大器：在一个反相放大器，输出信号将输入信号的倒版，是由某些因素放大。 4，仪表放大器：这是一个类型的差分放大器输入额外的缓冲阶段。输入阻抗高，易于匹配结果。仪表放大器具有更好的稳定性，高共模抑制比(CMRR)，低失调电压和高增益。
测量相位差的主要方法
一二测量相位差的方法主要有哪些？ 测量相位差可以用示波器测量，也可以把相位差转换为时间间隔，先测量出时间间隔，再换算为相位差，可以把相位差转换为电压，先测量出电压，再换算为相位差，还可以与标准移相器进行比较的比较法(零示法)等方法。 怎么用示波器来测量相位差? 应用示波器测量两个同频正弦电压之间的相位差的方法很多，本节介绍具有实用意义的直接比较法。将u1、u2分别接到双踪示波器的Y1通道和Y2通道，适当调节扫描旋钮和Y增益旋钮，使荧光屏显示出如图2.42所示的上、下对称的波形。 比较法测量相位差 设u1过零点分别为A、C点，对应的时间为t A、t C；u2过零点分别为B、D点，对应的时间为t B、t D。正弦信号变化一周是360°，u1过零点A比u2过零点B提前t B-t A出现，所以u1超前u2的相位。 u1超前u2的相位，即u1与u2的相位差为 (2.56) T为两同频正弦波的周期; ΔT为两正弦波过零点的时间差。 数字式相位计的结构与工作原理是什么?
三数字相位计框图 将待测信号u1(t)和u2(t)经脉冲形成电路变换为尖脉冲信号，去控制双稳态触发电路产生宽度等于ΔT的闸门信号以控制时间闸门的启、闭。晶振产生的频率为fc的正弦信号，经脉冲形成电路变换成频率为fc的窄脉冲。 在时间闸门开启时通过闸门加到计数器， 得计数值n，再经译码，显示出被测两信号的相位差。这种相位计可以测量两个信号的“瞬时”相位差，测量迅速，读数直观、清晰。 数字式相位计称做“瞬时”相位计，它可以测量两个同频正弦信号的瞬时相位，即它可以测出两同频正弦信号每一周期的相位差。 基于相位差转换为电压方法的模拟电表指示的相位计的测量原理是什么? 如图2.44所示，利用非线性器件把被测信号的相位差转换为电压或电流的增量，在电压表或电流表表盘上刻上相位刻度，由电表指示可直读被测信号的相位差。转换电路常称做检相器或鉴相器。常用的鉴相器有差接式相位检波电路和平衡式相位检波电路两种。 数字相位计框 图
实验12 斜率鉴频与相位鉴频器
实验12 斜率鉴频与相位鉴频器 —、实验准备 1．做本实验时应具备的知识点： FM波的解调 斜率鉴频与相位鉴频器 2．做本实验时所用到的仪器： 变容二极管调频模块 斜率鉴频与相位鉴频器模块 双踪示波器 万用表 二、实验目的 1．了解调频波产生和解调的全过程以及整机调试方法，建立起调频系统的初步概念； 2．了解斜率鉴频与相位鉴频器的工作原理； 3．熟悉初、次级回路电容、耦合电容对于电容耦合回路相位鉴频器工作的影响。 三、实验内容 1．调频-鉴频过程观察：用示波器观测调频器输入、输出波形，鉴频器输入、输出波形； 2．观察初级回路电容、次级回路电容、耦合电容变化对FM波解调的影响。 四、基本原理 从FM信号中恢复出原基带调制信号的技术称为FM波的解调，也称为频率检波技术，简称鉴频。鉴频器的解调输出电压幅度应与输入FM波的瞬时频率成正比，因此鉴频器实际上是一个频率—电压幅度转换电路。实现鉴频的方法有很多种，本实验介绍斜率鉴频和电容耦合回路相位鉴
频。 1．斜率鉴频电路 斜率鉴频技术是先将FM波通过线性频率振幅转换网络，使输出FM波的振幅按照瞬时频率的规律变化，而后通过包络检波器检出反映振幅变化的解调信号。实践中频率振幅转换网络常常采用LC并联谐振回路，为了获得线性的频率幅度转换特性，总是使输入FM波的载频处在LC并联回路幅频特性曲线斜坡的近似直线段中点，即处于回路失谐曲线中点。这样，单失谐回路就可以将输入的等幅FM波转变为幅度反映瞬时频率变化的FM波，而后通过二极管包络检波器进行包络检波，解调出原调制信号以完成鉴频功能。 图12-1为斜率鉴频与相位鉴频实验电路，图中13K02开关打 向“3”时为斜率鉴频。13Q01用来对FM波进行放大，13C2、13L02为频率振幅转换网络，其中心频率为9MHZ左右。13D03为包络检波二极管。13TP01、13TP02为输入、输出测量点。 2．相位鉴频器 本实验采用平衡叠加型电容耦合回路相位鉴频器，实验电路如图12-1所示，开关13K02拨向“1”时为相位鉴频。 相位鉴频器由频相转换电路和鉴相器两部分组成。输入的调频信号加到放大器13Q01的基极上。放大管的负载是频相转换电路，该电路是通过电容13C3耦合的双调谐回路。初级和次级都调谐在中心频率上。初级回路电压直接加到次级回路中的串联电容13C04、13C05的中心点上，作为鉴相器的参考电压；同时，又经电容13C3耦合到次级回路，作为鉴相器的输入电压，即加在13L02两端用表示。鉴相器采用两个并联二极管检波电路。检波后的低频信号经RC滤波器输出。
电压比较器原理及使用
实验十电压比较器的安装与测试 一．实验目的 1．了解电压比较器的工作原理。 2．安装和测试四种典型的比较器电路：过零比较器、电平检测器、滞回比较器和窗口比较器。 二．预习要求 1．预习过零比较器、电平检测器、滞回比较器和窗口比较器的工作原理。 2．预习使用示波器测量信号波形和电压传输特性的方法。 三．实验原理 电压比较器的基本功能是能对两个输入电压的大小进行比较，判断出其中那一个比较大。比较的结果用输出电压的高和低来表示。电压比较器可以采用专用的集成比较器，也可以采用运算放大器组成。由集成运算放大器组成的比较器，其输出电平在最大输出电压的正极限值和负极限值之间摆动，当要和数字电路相连接时，必须增添附加电路，对它的输出电压采取箝位措施，使它的高低输出电平，满足数字电路逻辑电平的要求。 下面讨论几种常见的比较器电路。 基本过零比较器（零电平比较器） 过零比较器主要用来将输入信号与零电位进行比较，+15V 以决定输出电压的极性。电路如图1所示：u i 2 7 放大器接成开环形式，信号u i从反向端输入，同μA7416u o 相端接地。当输入信号u i< 0时，输出电压u o为正极限34 值U OM；由于理想运放的电压增益A u→∞，故当输-15V 入信号由小到大，达到u i = 0 时，即u -= u + 的时刻， 输出电压u o 由正极限值U OM 翻转到负极限值-U OM。图 1 反向输入过零比较器 当u i >0时输出u o为负极限值-U OM。因此，输出翻转的临界条件是u + = u - = 0。 即：+U OM u i< 0 u o = （1） -U OM u i >0 其传输特性如图2（a）所示。所以通过该电路输出的电压值，就可以鉴别输入信号电压u i是大于零还是小于零，即可用做信号电压过零的检测器。
(相位鉴频器)电子测量实验指导书(科)
Xb08610209 陆斌 08电子信息（2）班 相位鉴频器 一、实验目的 1、熟悉相位鉴频电路的基本原理。 2、了解鉴频特性曲线（S 曲线）的正确调整方法。 3、将变容二极管调频器与相位鉴频器两实验板进行联机调试，进一步了解调频和解调全过程及整机调试方法。 二、实验原理 相位鉴频器是模拟调频信号解调的一种最基本的解调电路，它具有鉴频灵敏度高，解调线性好等优点。 1、鉴频概述 调频波的解调称为频率解调，简称鉴频；调相波的解调称为相位检波，简称 鉴相。它们的作用都是从已调波中检出反映在频率或相位变化上的调制信号。但是采用的方法不尽相同。由于在调频接收机中，当等幅调频信号通过鉴频前各级电路时，因电路频率特性不均匀而导致调频信号频谱结构的变化，从而造成调频信号的振幅发生变化。如果存在着干扰，还会进一步加剧这种振幅的变化。鉴频器解调这种信号时，上述寄生调幅就会反映在输出解调电压上，产生解调失真。因此，一般必须在鉴频前加一限幅器以消除寄生调幅，保证加到鉴频器上的调频电压是等幅的。限幅与鉴频一般是连用的，统称为限幅鉴频器。 鉴频器输出电压u 0随输入频率f （或频偏 ）变化的特性称为鉴 频特性。在线性解调的理想情况下，鉴频特性为一直线，实际上会弯曲，呈“S”型，称为“S”曲线。 2、鉴频器指标 1）鉴频跨导（效率、灵敏度）S D ：鉴频特性在f c 处的斜率，用它来评价鉴频能力。 单位为V/Hz 。S D 越大，表明鉴频器将输入瞬时频偏变换为输出解调电压的能力越强。 c f f f -=?
一般情况下，S D 为调制角频率的复值函数，即()D S j Ω，要求它的通频带大于调制信号的最高频率 m ax Ω 2）峰值带宽max B ：鉴频器输出电压两峰值点所对应的频率差，即 max 21B f f =-，它近似表明鉴频器鉴频线性区的宽度。为了减小鉴频器的非线性 失真，要求鉴频特性近似线性的范围 m ax 2f ?大于2m f ?。 ③ 最大输出电压0m ax U ：鉴频器输出的最大电压。 ④ 线性度要好与失真要小。 3.电容耦合双调谐回路相位鉴频器： 相位鉴频器的组成方框图如3-3示。图中的线性移相网络就是频—相变换网络，它将输入调频信号u1 的瞬时频率变化转换 为相位变化的信号u2，然后与原输入的调频信号一起加到相位检波器，检出反映频率变化的相位变化，从而实现了鉴频的目的。 图3-4的耦合回路相位鉴频器是常用的一种鉴频器。这种鉴频器的相位检波器部分是由两个包络检波器组成，线性移相网络采用耦合回路。为了扩大线性鉴频的范围，这种相位鉴频器通常都接成平衡和差动输出。 图3-4 耦合回路相位鉴频器 图3-5（a ）是电容耦合的双调谐回路相位鉴频器的电路原理图，它是由调 o
多序列比对的可视化显示
【一】多序列比对的可视化显示 可能因为毕业论文内容论文需要，最近很多人都找我帮忙将clustal的序列比对文件结果可视化，现将TEXshade软件包能做出来的可视化效果分享给各位同学，因为使用TEXshade 涉及到了一些LATEX知识，所以需要更深入的了解如何运作请给我留言或者私聊，此文仅将软件能做出的效果展示，虽然其实软件很简单，但不做具体使用说明，我的风格通常是授人以鱼，不授人以渔，嘻嘻。 最简单的莫过于纯粹的多序列比对排版，跟某些同学论文里面用Word排版的效果类似（word 里面需要用等宽字体），效果类似如下： 如果稍微加点效果可能就变成了如下的样子，我们将相同的氨基酸标记出来： 当然，TEXshade能做的不仅仅是这些，下面这张图我们标记了其中的几个关键位置，去掉了右侧的“ruler”
好像这个样子就有点乱了 下面这幅就更详细一点了，我用不同的颜色代表了不同的conservation
很多情况下，我们只想呈现多序列中的某些突变位点，其实也可以很好的表达 如果将T-Coffee 的 score_ascii 文件一并输入TEXshade，效果我觉得很帅气 我遇到过很多可视化表达各种结构域的例子，下面的表示是不是很帅呢？
下面是另外的一些功能实例 下面的被称为Sequence fingerprints，其实加上fingerprint命令就可以直接出图了。
很早的时候，看到序列的LOGO图觉得很帅气，下面的Logo图你见过么？
下面再给大家分享几个例子，其实TEXshade能做的很多，要靠大家的想象，我一向认为，只要人能想得出，数据可视化就能做得到。 所属相册：数据可视化
相位鉴频器
课程名称通信电子线路 实验项目相位鉴频器成绩 学院信息专业通信工程学号20141060149姓名李越 实验时间2016.06.04实验室3501指导教师谢汝生 1.实验目的 1.熟悉变容二级管调频器和相位鉴频器电路原理及构成。 2.了解调频器调制特性和相位鉴频器的鉴相特性及测量方法。 3.将变容二极管调频器与相位鉴频器两实验板进行联机试验，进一步了解调 频和解调全过程及整机调试方法。 2.实验设备 1.双踪示波器（RIGOL DS5062CA数字存储示波器） 2.频率计（AT-F1000-C数字频率计） 3.万用表（DT9205数字万用表） 4.扫频仪（BT3C宽带扫频仪）
5.清华科教TPE-GP2型高频电路实验箱及G4实验板 6.高频信号发生器（前锋QF1055A/1056A信号发生器） 3.实验电路及基本原理分析 从调频波中取出原来的调制信号，称为频率检波，又称为鉴频。在调频波中，调制信号包含在高频振荡频率的变化量中，所以调频波的解调任务就是要求鉴频器输出信号与输入调频波的瞬时频移成线性关系。 鉴频器电路是先借助谐振电路将等幅的调频波转换为幅度随瞬时频率变化的调幅调频波，再用二极管检波器进行幅度检波，以还原出调制信号。由于信号的最后检出还是利用高频振幅的变化，为了避免寄生调幅干扰检出的调制信号，一般都将输入鉴频器的调频波进行限幅去干扰，使其幅度恒定后再进行鉴频。
相位鉴频器是利用回路的相位-频率特性来实现调频波变换为调幅调频波的。它是将调频信号的频率变化转换为两个电压之间相位变化，再将这相位变化转换为对应的幅度变化，然后利用幅度检波器检出幅度变化。 本实验所用电路如图，该电路为电容耦合回路叠加型相位鉴频器。电路中V1/V2构成差分对振幅限幅电路，对输入信号进行去干扰限幅。同时在V2的集电极负载回路中设置了由CT1、C6、L1组成的并联谐振回路，与由CT2、C10、i 为调幅调频波。再通过后面两只检波二极管D1、D2组成的对称幅度检波器分别对上下两个调幅包络进行检波，最后得到调制信号。 4.实验步骤及内容记录（包括数据、图表、波形、程序设计等） 1.用扫频仪调整相位鉴频器的S型鉴频特性。 将实验电路中E、F、G三个接点分别与半可调电容C T1、C T2、C T3连接。
运放与比较器的用法
运放与比较器的用法 NE5532是双极型双运放，剩下的一个不用，可以将它们的输入端全部悬空即可，跟该运放相似的还有LM358、LM324，它们不用的输入端都可以悬空。而对于CMOS运放，由于输入阻抗极高，若将输入端悬空，很容易受干扰，故对于像ICL7642、MC14573这类CMOS 运放，内部用不完的运放，输入端一般要接高电平或地。 1、运放可以连接成为比较输出，比较器就是比较。 2、比较器输出一般是OC，便于电平转换；比较器没有频补，Slew Rate比同级运放大， 但接成放大器易自激。 比较器的开环增益比一般放大器高很多,因此比较器正负端小的差异就引起输出端变化. 3、频响是一方面，另外运放当比较器时输出不稳定，不一定能满足后级逻辑电路的要求。 4、比较器为集电极开路输出，容易输出TTL电平，而运放有饱和压降，使用不便。 关于运算放大器与专用比较器的区别可分为以下几点: 1.比较器的翻转速度快,大约在ns数量级,而运放翻转速度一般为us数量级(特殊高速运放 除外); 2.运放输入可以接成负反馈电路,而比较器不能使用负反馈,虽然比较器也有同相和反相两 个输入端,但因为其内部没有相位补偿电路,如果输入负反馈,电路不能稳定工作,内部无相位补偿电路.这也是比较器比运放速度快的原因. 3.运放的初级一般采用推挽电路,双极性输出,而多数比较器输出极为集电级开路结构,所 以需要上拉电阻,单极性输出,容易和数字电路连接. 加法器和减法器就是用运算放大器搭的运算电路. 电压比较器 电压比较器可以看作是放大倍数接近―无穷大‖的运算放大器。 电压比较器的功能：比较两个电压的大小(用输出电压的高或低电平，表示两个输入电压的大小关系)： 当‖＋‖输入端电压高于‖－‖输入端时，电压比较器输出为高电平； 当‖＋‖输入端电压低于‖－‖输入端时，电压比较器输出为低电平； 电压比较器的作用：它可用作模拟电路和数字电路的接口，还可以用作波形产生和变换电路等。利用简单电压比较器可将正弦波变为同频率的方波或矩形波。 简单的电压比较器结构简单，灵敏度高，但是抗干扰能力差，因此我们就要对它进行改进。改进后的电压比较器有：滞回比较器和窗口比较器。 运放，是通过反馈回路和输入回路的确定―运算参数‖，比如放大倍数，反馈量可以是输出的电流或电压的部分或全部。而比较器则不需要反馈，直接比较两个输入端的量，如果同相输入大于反相，则输出高电平，否则输出低电平。电压比较器输入是线性量，而输出是开关（高低电平）量。一般应用中，有时也可以用线性运算放大器，在不加负反馈的情况下，构成电压比较器来使用。
高频实验九 电容耦合相位鉴频器实验报告
实验九 电容耦合相位鉴频器实验 一．实验目的 1. 进一步学习掌握频率解调相关理论。 1. 了解电容耦合回路相位鉴频器的工作原理。 3. 了解鉴频特性（S 形曲线的调试与测试方法）。 二、实验使用仪器 1．电容耦合相位鉴频器实验板 2．100MH 泰克双踪示波器 3. FLUKE 万用表 4. 高频信号源 三、实验基本原理与电路 1. 实验基本原理 从调频波中取出原来的调制信号，称为频率检波，又称鉴频。完成鉴频功能的电路，称为鉴频器。在调频波中，调制信息包含在高频振荡频率的变化量中，所以调频波的解调任务就是要求鉴频器输出信号与输入调频波的瞬时频移成线性关系。 本实验采用的是相位鉴频器。相位鉴频器是利用回路的相位-频率特性来实现调频波变换为调幅调频波的。它是将调频信号的频率变化转换为两个电压之间的相位变化，再将这相位变化转换为对应的幅度变化，然后利用幅度检波器检出幅度的变化。 鉴相器采用两个并联二极管检波电路。假设二极管D3的检波电路和二极管D4的检波电路完全对称，两个检波电路的电压传输系数完全相等，检波后的输出信号为两个检波电路的输出电压差。即034D D U U U =- 当瞬时频率0f f =时, 2U 比1U 滞后90°，但|3D U |＝|4D U |，这时，鉴频器输出为零。当0f f >时, 2U 滞后于1U 的相角小于90°，|3D U |>|4D U |，鉴频器的输出大于零。当0f f <时，2U 滞后于1U 的相角大于90°，
|3D U |<|4D U |，鉴频器的输出小于零。相位鉴频器鉴频特性的线性较好，鉴频灵敏度也较高。 图9-1频率电压转换原理图。 (ω<ω0)U 2(ω=ω0) (ω>ω0) . U 1.. U 2 .2U 2. 2 .. U 1 .U 2 .2 U 2. 2 . . U 2 .2 U 2. 2 (a) (b)(ω=ω0)(c)(ω>ω0) (d)(ω<ω0) 图9-1频率电压转换原理图。 鉴频器的主要参数： （1） 鉴频跨导 鉴频器的输出电压与输入调频波的瞬时频率偏移成正比，其比例系数称为鉴频跨导。图9-3为鉴频器输出电压V 与调频波的瞬时频偏f ?之间的关系曲线，称为鉴频特性曲线。它的中部接近直线部分的斜率即为鉴频跨导。它代表每单位频偏所产生的输出电压的大小，希望鉴频器的鉴频跨导应该尽可能的大。 （2）鉴频灵敏度 指鉴频器正常工作时，所需要输入调频波的最小幅度。其值越小，鉴频器灵敏度越高。 （3）鉴频器频带宽度 从上图的鉴频特性曲线中可以看出，只有特性曲线中间一部分的线性度较好，我们称2m f ?为频带宽度。一般，要求2m f ?大于输入调频波频偏的两倍，并
运算放大器可以用作比较器
运算放大器可以用作比较器 许多人偶尔会把运算放大器当比较器使用。一般而言，当您只需要一个简单的比较器，并且您在四运算放大器封装中还有一个“多余”的运算放大器时，这种做法是可行的。只是运算放大器需要相位补偿才能运行，因而把运算放大器用作比较器时其速度会非常低，但是如果对速度要求不高，则运算放大器可以满足需求。偶尔会有人问到我们运算放大器的这种使用方法，因为他们发现这种方法有时有效，有时却不如人们预期的那样效果好。为什么会出现这种情况呢？ 许多运算放大器都在输入端之间有电压钳位，其大多数一般都使用背靠背二极管（有时使用两个或者更多的串联二极管）来实施。这些二极管保护输入晶体管免受其基极结点反向击穿的损害。许多IC工艺在差动输入约为6V时便会出现击穿，这会极大地改变或者损坏晶体管。图1显示了NPN输入级，D1和D2提供了这种保护功能。 图1 在大多数常见运算放大器应用中，输入电压均约为零伏，根本无法开启这些二极管。但是很明显，对于比较器的运行而言，这种保护便成了问题。在一个输入拖拽另一个输入（以一种讨厌的方式拉其电压）以前，差动电压范围（约0.7V）受限。尽管如此，我们还是可以把运算放大器用作比较器。但是，在我们这样做时必须小心谨慎。在一些电路中，这种做法可能是完全不能接受的。问题是我们（包括其他运算放大器厂商）并没有总是说明这些钳位的存在，即使有所说明，可能也不会做详细的解释或
者阐述。也许我们应该说：“用作比较器时，请小心谨慎！”产品说明书的作者们通常也只是假设您肯定会把运算放大器当作运算放大器用。 TI在美国亚利桑那州图森产品部召开了一个会议，会议决定，TI以后将会更加清楚地说明这种情况。但是，现在已经生产出来的运算放大器怎么办呢？下列指导建议可能会对您有所帮助： 一般而言，双极NPN晶体管运算放大器都有输入钳位，例如：OP07、OPA227和 OPA277等。uA741是一个例外，它具有NPN输入晶体管，并且有一些为NPN提供固有保护的附加串联横向PNP。 图2 使用横向PNP输入晶体管的通用运算放大器一般没有输入钳位，例如：LM324、LM358、OPA234、OPA2251和OPA244。这些运算放大器一般为“单电源”类型，意味着它们拥有扩展至负电源端（或者稍低）的共模范围。输入偏置电流为负数时，表示输入偏置电流自输入引脚流出。这时，通常可以认定它们为这类运算放大器。但是，需要注意的是，使用PNP输入的高速运算放大器一般有输入钳位，而这些PNP是一些具有更低击穿电压的垂直PNP。
高精度测相技术比较
高精度测相方法的研究和比较 摘要：相位测量技术的研究由来已久，最早的研究和应用是在数学的矢量分析和物理学的圆周运动以及振动学方面，随之在电力部门、机械部门、航空航天、地质勘探、海底资源等方面也相应得到重视和发展。随着电子技术和计算机技术的发展，相位测量技术得到了迅速的发展。本文主要阐述常用相位测量技术和数字测相，介绍这两种方法里面的一些具体测量手段并做了详细研究和比较。 关键词：常用相位测量技术；数字测相； The Study and Compare of Phase Measurement Technology WEI Kaiming, LIU Zhihao (University of Electronic Science and Technology of China, School of Automation Engineering) Abstract: Phase measurement technology research for a long time, is the earliest research and application of vector analysis in mathematics and the circular motion of physics science and vibration. Then in the electric power department, the department of mechanical, aerospace, geological exploration, undersea resources also pay attention to and develop corresponding. With the development of electronic technology and computer technology, Phase measurement technology got rapid development. This article mainly elaborates common phase measurement technology and digital phase, introduces the two methods in some specific measures and made a careful study and comparison. Key words:Common phase measurement technology；Digital phase measurement；Comparison 现代相位测量技术的发展可分为三个阶段:第一阶段是在早期采用的诸如阻抗法、和/差法、三电压法、比对法和平衡法等,虽然方法简单,但测量精度较低；第二阶段是利用数字专用电路、微处理器、FPGA、CPLD、DSP等构成测相系统,使测量精度得以大大提高；第三阶段是充分利用计算机及智能化虚拟测量技术,从而大大简化设计程序,增强功能,使得相应的产品精度更高、功能更全。 与此同时,各种新的算法也随之出现。目前国内外各种新的测量算法、测量手段和新的设计方法及器件也随之出现，主要包括有:用专用数字处理芯片,利用正余弦表格及傅立叶变换方法来计算相位差,可以大大提高测量精度。采用新器件及设计方法提高相位测量精度及展宽
乘积型相位鉴频器的设计
课程设计任务书 学生姓名：专业班级： 指导教师：工作单位： 题目：乘积型相位鉴频器的设计 初始条件： 具较扎实的电子电路的理论知识及较强的实践能力；对电路器件的选型及电路形式有一定的了解；具备晶体管电路的基本设计及基本调试能力；能够正确使用实验仪器进行电路的调试与检测；使用适当的软件进行仿真和制作PCB板图。 主要内容： 本题目为集成模拟乘法器应用设计之一，即设计一个乘积型相位鉴频器。通过本次电路设计，掌握集成模拟乘法器的基本原理及其所构成的相位鉴频电路的设计方法、电路调整及测试技术。加深对高频电子线路课程理论知识的理解，提高电路设计及电子实践能力。 基本要求： (1) 采用集成模拟乘法器设计乘积型相位鉴频器，电路的工作中心频率 为f=6.5MHz。 (2) 绘制电路原理图，并给出鉴频特性曲线。 指导教师签名：年月日 系主任（或责任教师）签名：年月日
目录 1 原理说明与电路分析 (3) 1.1电路原理及用途.........................................................................................错误！未 定义书签。 2.2 模拟乘法器MC1496 (4) 2.3 低通滤波器 (5) 2.4主要技术指标 (5) 3 乘积型相位鉴频器 (8) 3.1 乘积型相位鉴频器的原理图....................................................................错误！未 定义书签。 3.2电路工作状态或元件参数的确定 (9) 3.3仿真结果 (11) 3.4 调试及静态工作点的测量 (14) 4 元件清单 (16) 5 心得体会 (17) 6参考文献 (18)