Article Figures & Data
Figures
- Fig. 1.
Schematic illustration of proteome analysis by 2DE and mass spectrometry. In part I, proteins are separated by 2DE, stained spots are excised and subjected to in-gel digestion with trypsin, and the resulting peptides are separated by on-line capillary high-performance liquid chromatography. In part II, a peptide is shown eluting from the column in part I. The peptide is ionized by electrospray ionization and enters the mass spectrometer. The mass of the ionized peptide is detected, and the first quadrupole mass filter allows only the specific mass-to-charge ratio of the selected peptide ion to pass into the collision cell. In the collision cell, the energized, ionized peptides collide with neutral argon gas molecules. Fragmentation of the peptide is essentially random but occurs mainly at the peptide bonds, resulting in smaller peptides of differing lengths (masses). These peptide fragments are detected as a tandem mass (MS/MS) spectrum in the third quadrupole mass filter where two ion series are recorded simultaneously, one each from sequencing inward from the N and C termini of the peptide, respectively. In part III, the MS/MS spectrum from the selected, ionized peptide is compared to predicted tandem mass spectra computer generated from a sequence database. Provided that the peptide sequence exists in the database, the peptide and, by association, the protein from which the peptide was derived can be identified. Unambiguous protein identification is attained in a single analysis because multiple peptides are identified as being derived from the same protein.
- Fig. 2.
2D silver-stained gel of the proteins in yeast total cell lysate. Proteins were separated in the first dimension (horizontal) by isoelectric focusing and then in the second dimension (vertical) by molecular weight sieving. Protein spots (156) were chosen to include the entire range of molecular weights, isoelectric focusing points, and staining intensities. Spots were excised, and the corresponding protein was identified by mass spectrometry and database searching. The spots are labeled on the gel and correspond to the data presented in Table 1. Molecular weights are given in thousands.
- Fig. 3.
Tandem mass (MS/MS) spectra resulting from analysis of a single spot on a 2D gel. The first quadrupole selected a single mass-to-charge ratio (m/z) of 687.2 (A) or 592.6 (B), while the collision cell was filled with argon gas, and a voltage which caused the peptide to undergo fragmentation by CID was applied. The third quadrupole scanned the mass range from 50 to 1,400 m/z. The computer program Sequest (8) was utilized to match MS/MS spectra to amino acid sequence by database searching. Both spectra matched peptides from the same protein, S57593 (yeast hypothetical protein YMR226C). Five other peptides from the same analysis were matched to the same protein.
- Fig. 4.
Current proteome analysis technology utilizing 2DE without preenrichment samples mainly highly expressed and long-lived proteins. Genes encoding highly expressed proteins generally have large codon bias values. (A) Distribution of the yeast genome (more than 6,000 genes) based on codon bias. The interval with the largest frequency of genes is 0.0 to 0.1, with more than 2,500 genes. (B) Distribution of the genes from identified proteins in this study based on codon bias. No genes with codon bias values less than 0.1 were detected in this study. (C) Distribution of identified proteins in this study based on predicted half-life (estimated by N-end rule).
- Fig. 5.
Correlation between protein and mRNA levels for 106 genes in yeast growing at log phase with glucose as a carbon source. mRNA and protein levels were calculated as described in Materials and Methods. The data represent a population of genes with protein expression levels visible by silver staining on a 2D gel chosen to include the entire range of molecular weights, isoelectric focusing points, and staining intensities. The inset shows the low-end portion of the main figure. It contains 69% of the original data set. The Pearson product moment correlation for the entire data set was 0.935. The correlation for the inset containing 73 proteins (69%) was only 0.356.
- Fig. 6.
Effect of highly abundant proteins on Pearson product moment correlation coefficient for mRNA and protein abundance in yeast. The set of 106 genes was ranked according to protein abundance, and the correlation value was calculated by including the 40 lowest-abundance genes and then progressively including the remaining 66 genes in order of abundance. The correlation value climbs as the final 11 highly abundant proteins are included.
- Fig. 7.
Relationship between codon bias and protein and mRNA levels in this study. Yeast mRNA and protein expression levels were calculated as described in Materials and Methods. The data represent the same 106 genes as in Fig. 5.
Tables
- Table 1.
Expressed genes identified from 2D gel in Fig. 2
Mol wt pI Spot no. YPD gene namea Protein abundance (103copies/cell) mRNA abundance (copies/cell) Codon bias 17,259 6.75 133 CPR1 15.2 61.7 0.769 18,702 4.80 83 EGD2 20.1 5.2 0.724 18,726 4.44 147 YKL056C 61.2 88.4 0.831 18,978 5.95 135 YER067W 3.7 6.7 0.118 19,108 5.04 130 YLR109W 94.4 9.7 0.680 19,681 9.08 136 ATP7 11.0 NAb,c 0.246 20,505 6.07 111 GUK1 16.5 3.7 0.422 21,444 5.25 148 SAR1 5.4 10.4 0.455 21,583 4.98 95 TSA1 110.6 40.1 0.845 22,602 4.30 80 EFB1 66.1 23.8 0.875 23,079 6.29 112 SOD2 12.6 2.2 0.351 23,743 5.44 137 HSP26 NAd 0.7 0.434 24,033 5.97 96 ADK1 17.4 16.4 0.656 24,058 4.43 143 YKL117W 29.2 10.4 0.339 24,353 6.30 140 TFS1 8.1 0.7 0.146 24,662 5.85 99 URA5 25.4 6.0 0.359 24,808 6.33 97 GSP1 26.3 5.2 0.735 24,908 8.73 122 RPS5 18.6 NAc 0.899 25,081 4.65 81 MRP8 9.3 NAc 0.241 25,960 6.06 116 RPE1 5.8 0.7 0.372 26,378 9.55 127 RPS3 96.8 NAc 0.863 26,467 5.18 100 VMA4 10.5 3.7 0.427 26,661 5.84 98 TPI1 NAd NAc 0.900 27,156 5.56 93 PRE8 6.9 0.7 0.129 27,334 6.13 115 YHR049W 18.4 2.2 0.520 27,472 5.33 92 YNL010W 31.6 3.7 0.421 27,480 8.95 123 GPM1 10.0 169.4 0.902 27,480 8.95 124 GPM1 231.4 169.4 0.902 27,480 8.95 125 GPM1 7.5 169.4 0.902 27,809 5.97 139 HOR2 5.7 0.7 0.381 27,874 4.46 78 YST1 13.6 52.8 0.805 28,595 4.51 41 PUP2 4.4 0.7 0.147 29,156 6.59 114 YMR226C 14.5 2.2 0.283 29,244 8.40 120 DPM1 5.0 11.2 0.362 29,443 5.91 48 PRE4 3.4 3.7 0.162 30,012 6.39 138 PRB1 21.2 1.5 0.449 30,073 4.63 77 BMH1 14.7 28.2 0.454 30,296 7.94 121 OMP2 67.4 41.6 0.499 30,435 6.34 89 GPP1 70.2 11.2 0.703 31,332 5.57 88 ILV6 13.9 3.0 0.402 32,159 5.46 113 IPP1 63.1 3.7 0.752 32,263 6.00 149 HIS1 22.4 4.5 0.232 33,311 5.35 84 SPE3 15.1 6.7 0.468 34,465 5.60 129 ADE1 8.7 5.2 0.305 34,762 5.32 85 SEC14 10.9 6.0 0.373 34,797 5.85 42 URA1 49.5 8.9 0.237 34,799 6.04 90 BEL1 103.2 81.0 0.875 35,556 5.97 43 YDL124W 6.4 4.5 0.206 35,619 8.41 59 TDH1 69.8 32.7c 0.940 35,650 5.49 68 CAR1 5.2 3.0 0.339 35,712 6.72 117 TDH2 49.6 473.0c 0.982 35,712 6.72 154 TDH2 863.5 473.0c 0.982 35,712 6.72 155 TDH2 79.4 473.0c 0.982 36,272 4.85 128 APA1 8.7 0.7 0.425 36,358 5.05 75 YJR105W 17.6 17.1 0.522 36,358 5.05 76 YJR105W 27.5 17.1 0.522 36,596 6.37 79 ADH2 58.9 260.0c 0.711 36,714 6.30 102 ADH1 746.1 260.0 0.913 36,714 6.30 103 ADH1 17.6 260.0 0.913 36,714 6.30 104 ADH1 61.4 260.0 0.913 36,714 6.30 105 ADH1 52.7 260.0 0.913 37,033 6.23 44 TAL1 44.8 3.7 0.701 37,796 7.36 57 IDH2 29.4 6.7 0.330 37,886 6.49 106 ILV5 76.0 4.5 0.892 38,700 7.83 55 BAT1 30.9 11.2 0.469 38,702 6.24 46 QCR2 NAd 2.2 0.326 39,477 5.58 86 FBA1 17.8 183.6 0.935 39,477 5.58 87 FBA1 427.2 183.6 0.935 39,540 6.50 150 HOM2 60.3 4.5 0.592 39,561 6.12 156 PSA1 96.4 27.5 0.718 41,158 6.01 49 YNL134C 14.9 1.5 0.316 41,623 7.18 58 BAT2 19.0 8.9 0.250 41,728 7.29 110 ERG10 24.1 4.5 0.543 41,900 5.42 74 TOM40 22.3 2.2 0.375 42,402 6.29 45 CYS3 6.7 8.9 0.621 42,883 5.63 67 DYS1 15.8 5.2 0.526 43,409 6.31 107 SER1 10.5 1.5 0.292 43,421 5.59 91 ERG6 2.2 14.1 0.408 44,174 7.32 56 YBR025C 13.1 6.0 0.684 44,682 4.99 72 TIF1 2.9 39.4 0.834 44,707 7.77 108 PGK1 23.7 165.7 0.897 44,707 7.77 109 PGK1 315.2 165.7 0.897 46,080 6.72 30 CAR2 15.4 NAc 0.495 46,383 8.52 53 IDP1 7.7 0.7 0.436 46,553 5.98 47 IDP2 32.4 NAc 0.197 46,679 6.39 50 ENO1 35.4 0.7 0.930 46,679 6.39 51 ENO1 6.6 0.7 0.930 46,679 6.39 52 ENO1 2.2 0.7 0.930 46,773 5.82 63 ENO2 15.5 289.1 0.960 46,773 5.82 64 ENO2 635.5 289.1 0.960 46,773 5.82 65 ENO2 93.0 289.1 0.960 46,773 5.82 66 ENO2 31.0 289.1 0.960 47,402 6.09 126 COR1 2.5 0.7 0.422 47,666 8.98 54 AAT2 11.7 6.0 0.338 48,364 5.25 73 WTM1 74.5 13.4 0.365 48,530 6.20 61 MET17 38.1 29.0 0.576 48,904 5.18 69 LYS9 16.2 3.7 0.463 48,987 4.90 153 SUP45 29.6 11.9 0.377 49,727 5.47 70 PRO2 13.6 5.2 0.297 49,912 9.27 62 TEF2 558.5 282.0 0.932 50,444 5.67 35 YDR190C 4.8 2.2 0.228 50,837 6.11 32 YEL047C 3.8 1.5 0.387 50,891 4.59 151 TUB2 11.2 7.4 0.404 51,547 6.80 27 LPD1 18.9 2.2 0.351 52,216 7.25 29 SHM2 19.7 7.4 0.722 52,859 5.54 37 YFR044C 30.2 6.7 0.442 53,798 5.19 71 HXK2 26.5 7.4 0.756 53,803 6.05 145 GYP6 4.4 0.7 0.147 54,403 5.29 39 ALD6 37.7 2.2 0.664 54,403 5.29 40 ALD6 6.6 2.2 0.664 54,502 6.20 31 ADE13 6.3 1.5 0.417 54,543 7.75 25 PYK1 225.3 101.8 0.965 54,543 7.75 26 PYK1 39.8 101.8 0.965 55,221 6.66 146 YEL071W 16.3 3.0 0.244 55,295 4.35 134 PDI1 66.2 14.1 0.589 55,364 5.98 24 GLK1 22.6 6.0 0.237 55,481 7.97 118 ATP1 21.6 2.2 0.637 55,886 6.47 28 CYS4 22.2 NAc 0.444 56,167 5.83 33 ARO8 14.3 3.0 0.324 56,167 5.83 34 ARO8 9.1 3.0 0.324 56,584 6.36 20 CYB2 18.9 NAc 0.259 57,366 5.53 60 FRS2 2.3 0.7 0.451 57,383 5.98 144 ZWF1 5.6 0.7 0.215 57,464 5.49 36 THR4 21.4 3.7 0.508 57,512 5.50 7 SRV2 6.5 NAc 0.260 57,727 4.92 152 VMA2 33.7 8.9 0.546 58,573 6.47 17 ACH1 4.4 1.5 0.327 58,573 6.47 18 ACH1 5.4 1.5 0.327 61,353 5.87 21 PDC1 6.5 200.7 0.962 61,353 5.87 22 PDC1 303.2 200.7 0.962 61,353 5.87 23 PDC1 16.3 200.7 0.962 61,649 5.54 38 CCT8 2.2 1.5 0.271 61,902 6.21 101 PDC5 4.3 NAc 0.828 62,266 6.19 16 ICL1 20.1 NAc 0.327 62,862 8.02 19 ILV3 5.3 4.5 0.548 63,082 6.40 119 PGM2 2.2 3.0 0.402 64,335 5.77 5 PAB1 30.4 1.5 0.616 66,120 5.42 8 STI1 6.7 0.7 0.313 66,120 5.42 9 STI1 6.4 0.7 0.313 66,450 5.29 141 SSB2 7.0 NAc 0.880 66,450 5.29 142 SSB2 2.3 NAc 0.880 66,456 5.23 10 SSB1 64.5 79.5 0.907 66,456 5.23 11 SSB1 59.0 79.5 0.907 66,456 5.23 12 SSB1 13.7 79.5 0.907 68,397 5.82 82 LEU4 3.1 3.0 0.407 69,313 4.90 13 SSA2 24.3 18.6 0.892 69,313 4.90 14 SSA2 77.1 18.6 0.892 74,378 8.46 15 YKL029C 2.8 3.7 0.353 75,396 5.82 6 GRS1 5.5 7.4 0.500 85,720 6.25 1 MET6 2.0 NAc 0.772 85,720 6.25 2 MET6 10.9 NAc 0.772 85,720 6.25 3 MET6 1.4 NAc 0.772 93,276 6.11 131 EFT1 17.9 41.6 0.890 93,276 6.11 132 EFT1 5.7 41.6 0.890 102,064e 6.61e 94 ADE3 4.8 5.2 0.423 107,482e 5.33e 4 MCM3 2.7 NAc 0.240 ↵a YPD gene names are available from the YPD website (39).
↵b NA, calculation could not be performed or was not available.
↵c mRNA data inconclusive or NA.
↵d No methionines in predicted ORF; therefore, protein concentration was not determined.
↵e Measured molecular weight or pI did not match theoretical molecular weight or pI.
