©2004 Waters Corporation ASMS 2004 Towards Global Proteomics by Analysis of Exact Mass Retention Time Pairs: A proof-of-principle of the Quantitative Ion Mapping Technology Scott J. Geromanos 1 ; Keith Richardson 1 ; Phillip Young 1 ; Richard Denny 1 ; Marc Gorenstein 1 ; Guo-Zhong Li 1 ; Timothy Riley 1 ; Jeffrey Silva 1 ; Gregory J. Opiteck 2 ; Ashok R. Dongre 2 ; and Stanley A. Hefta 2 1 Waters Corporation, Milford, MA; 2 Bristol-Myers Squibb, Pennington, NJ This poster illustrates the quantitative capabilities of the new Waters ® Protein Expression System. The data presented were generated using commercially available tryptically digested human serum samples spiked with different con- centrations of five exogenous internal reference proteins contained in the Wa- ters Protein Expression System Standards. Data were acquired in alternate scanning low and elevated energy accurate mass LC/MS mode. Each spiked digest sample was analyzed in triplicate. The raw data was than processed through a suite of Protein Expression System Informatics tools to produce a list of unique E xact M ass R etention-T ime (EMRT) components. The data are proc- essed further to match and compare the intensity of like EMRT components from different sample injections. Accurate mass low and elevated information associated with EMRT components that illustrate a significant change in inten- sity may optionally be exported to facilitate their qualitative identification. Human serum (Sigma source) was tryptically digested according to the procedure described in Poster TPY 458 (C. Dorschel et al., “Protocols to Assure Reproducible Quantitative and Qualitative Analysis of Tryptic Digests of Complex Protein Mixtures for Global Proteomic Experi- ments”). Five aliquots of this human serum digest were spiked with Waters Protein Expression System Standards, containing equimolar levels of Yeast Enolase and Alcohol Dehydrogenase, Rabbit Glycogen Phosphorylate, Bovine Serum Albumin and Hemoglobin tryptic digest. • LC/MS System: Waters Protein Expression System comprised of the Waters CapLC ® System with the Waters Micromass ® Q-Tof Ultima API Mass Spectrometer equipped with a NanoLockSpray™ Source operated at 12,000 mass resolving power • Column: Waters NanoEase™ Atlantis™ dC18 Column, 300 μm x 15 cm • Mobile Phase: A = 1% Acetonitrile in Water, 0.1% Formic Acid, B = 80% Acetonitrile in Water, 0.1% Formic Acid • Gradient: 6% to 40% B over 100 min. at 4.4 μL/min, followed by 10 min. rinse (99% B) and 20 min. re-equilibration at initial conditions Each of the five spiked human serum (HS) digest samples was analyzed in triplicate. Injections were 5 microliters each and were made directly on-column. The total protein load for each in- jection was ~8.5 micrograms of Human Serum plus either 5.0, 1.0, 0.5, 0.25 or 0.1 pico- moles of the spiked Protein Expression System Standards. Exact mass LC/MS data was col- lected using an alternating low (10eV) and elevated (28eV to 35eV) collision energy mode of acquisition such that one cycle of low and elevated collision energy data was acquired every 4.0 seconds (Figure 1). The NanoLockSpray source was switched every 10 seconds to obtain a reference scan of [Glu 1 ]-Fibrinopeptide lockmass calibrant. Figure 1. Data acquisition using the Waters Protein Expression System mass spectrometer All results illustrated in the Figures and Tables are based on data collected in the low collision energy portion of the low and elevated energy data acquisition cy- cle. Figure 2 panels A through O illustrate the base peak intensity (BPI) chroma- tograms of the triplicate analyses of each of the five spiked HS digest samples. Note that the appearance of all 15 BPI chromatograms is quite similar indicating that the spiked protein digest components represent only a minor fraction of the total peptide content of each HS digest mixture. Figure 3 panels A through O illustrate the peak area integration of a non- lockmass corrected, selected ion chromatogram of an example spiked peptide (mass 978.49 eluting at approximately 77.5 minutes). Figure 4 panels A through O illustrate the complexity of a single lock mass cor- rected spectrum extracted at the apex of each selected ion chromatogram. Figure 5 panels A through O show an expanded mass range region of each of these spectra centered on m/z 978.49. The complexity of the spectra and the presence of significantly higher intensity ions indicate that the 978.49 EMRT is a minor component eluting at that point in the chromatogram. The software has ex- tracted the 978.49 EMRT component with excellent reproducibility and its linear- ity in response across the various spiked injection levels indicates that ion sup- pression effects are minimal. The mass precision for all 15 measurements of the 978.49 EMRT is +/- 4.64 ppm. Figures 6 through 9 compare data extracted from the fifteen spiked HS injec- tions using the Protein Expression System Informatics Software. Figure 6B illus- trates the log ratio of EMRT components from the 5.0 and 0.1 picomole spiked HS samples. The cloud of ions lying along the 45 degree axis represents EMRT peptides that were measured with an intensity ratio of approximately 1.0. These peptides are associated with HS peptides. The methodology employed allowed these ions to be measured over nearly 4 orders of magnitude in intensity. The cloud of ions lassoed in the upper left section of panel b illustrates the EMRT pep- tides associated with the spiked exogenous protein digests. This intensity ratio in- formation is further illustrated in Figure 6C as a frequency plot. The intensity of the two EMRT groupings clearly illustrate the 1:1 ratio of the HS peptides and the 1:50 ratio of the spiked HS peptides. Figure 6A illustrates that the coefficient of variation of the intensity measurements made on the HS peptides was generally less than 20%. Figures 7 through 9 compare 5.0 picomole vs. 0.5 picomole spiked samples, 0.50 versus 0.25 picomole spiked samples, and 0.25 versus 0.1 picomole spiked samples, respectively. A method for finding significant changes in peptide intensity is through the use of a paired Student t-Test. This test was employed to test the intensity of all the EMRT peptides that were observed to be in at least 2 out of 3 replicate injections in all of the spiked samples. This analysis produced a list of 63 EMRT peptides that had an intensity ratios between samples with a p-score lower than 0.01(data not shown). Figure 10 shows typical concentration plots for 6 randomly selected peptides. A quick perusal shows linearity across three orders of magnitude on peptides of varying ionization efficiencies. This list of peptides was searched against the SwissProt database using 10 ppm mass accuracy search criteria. Table 2 illustrates that 44 of the 63 peptides (>67%) mapped back to the five exogenous proteins spiked into the HS digest samples. Figure 10. Concentration vs. Intensity Student t-Test Results Human Serum + 5 picomoles Waters Protein Expression System Standards Figure 6. 5 picomoles/100 femtomoles In Human Serum Figure 7. 5 picomoles/500 femtomoles In Human Serum Figure 8. 500 femtomoles/250 femtomoles In Human Serum Figure 9. 250 femtomoles/100 femtomoles In Human Serum Figure 2. BPI Chromatograms A B C Figure 3. SIC 978.xxxx D E F Human Serum + 1 picomole Standards G H I Human Serum + 0.5 picomoles Standards J K L Human Serum + 0.25 picomoles Standards M N O Human Serum + 0.1 picomoles Standards 600 800 1000 1200 m /z 0 100 % 080603_497HL_QuanExpSerial_07 869 (77.529) AM (Cen 2.22e4 790.9260 682.7010 669.8299 791.4247 820.8712 1045.9894 1046.4917 1046.9948 600 800 1000 1200 m /z 0 100 % 080603_497HL_QuanExpSerial_01 862 (76.861) AM (Cen 1.59e4 790.9269 682.7028 669.8300 791.4244 820.8719 1045.9910 1046.4923 1046.9946 600 800 1000 1200 m /z 0 100 % 080603_497HL_QuanExpSerial_13 868 (77.397) AM (Cen 2.34e4 790.9265 682.7001 791.4242 820.8721 821.3696 1045.9897 821.8726 1046.4911 1046.9940 977 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_07 869 (77.529) AM (Cen 3.72e3 978.9915 978.4916 977.5114 979.4922 979.9962 980.4995 977 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_01 862 (76.861) AM (Cen 2.90e3 978.9931 978.4935 977.5165 979.4935 979.9962 980.5021 977 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_13 868 (77.397) AM (Cen 4.02e3 978.9918 978.4919 977.4807 979.4923 979.9935 980.4962 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_03 868 (77.388) AM (Cen,4, 85.00, Ar,12000.0,7 1.16e4 820.8706 11559 682.7019 6038 682.3677 4011 683.0347 4309 821.3704 10147 821.8724 4900 937.4814 3465 847.8323 2010 1023.5581 3184 1131.0259 1036 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_09 869 (77.506) AM (Cen,4, 85.00, Ar,12000.0,7 1.89e4 682.7043 18903 682.3699 13883 669.8304 1592 683.0348 10713 820.8704 10078 683.3688 4385 937.9755 6199 821.8717 4237 1023.5587 5929 1024.0601 3862 1131.0311 605 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_15 873 (77.869) AM (Cen,4, 85.00, Ar,12000.0,7 1.33e4 820.8704 13251 682.7021 9377 682.3683 6462 628.2473 1074 683.0341 6139 821.3695 11558 821.8725 5308 937.9795 4841 847.8305 1770 1023.5575 3903 1131.0261 885 977 978 979 980 m/z 0 100 % 080603_497HL_QuanExpSerial_03 869 (7 727 978.4924 977.5117 977.9971 978.9933 979.4988 979.9883 977 978 979 980 m/z 0 100 % 080603_497HL_QuanExpSerial_09 869 (7 689 978.9965 978.4922 977.5118 978.0760 979.4936 979.9967 977 978 979 980 m/z 0 100 % 080603_497HL_QuanExpSerial_15 874 (7 851 978.9916 978.4910 977.5134 977.9865 979.4878 979.9998 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_04 870 (77.611) AM (Cen,4, 85.00, Ar,12000 2.92e4 682.7063 682.3712 669.8302 683.0350 1023.5591 820.8697 683.3691 683.7068 937.9747 821.8708 938.9804 1024.0618 1024.5626 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_10 867 (77.291) AM (Cen,4, 85.00, Ar,12000 1.42e4 682.7024 682.3688 669.8305 820.8701 683.0345 683.3688 683.7063 1023.5593 937.9771 1024.0610 1024.5647 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_16 878 (78.312) AM (Cen,4, 85.00, Ar,12000 3.81e4 682.7018 669.8304 683.0366 1023.5592 937.9731 683.3690 820.8691 938.9786 1024.0597 1024.5624 1131.0239 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_04 870 388 978.4943 978.9926 979.4992 979.9919 980.4755 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_16 878 339 978.4979 978.9942 979.4996 980.4976 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_10 867 286 978.5023 978.9941 979.4996 979.9974 980.9815 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_06 876 (78.0 132 978.4932 132 979.5203 96 979.0084 72 980.4995 50 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_12 862 (76.8 131 978.5094 131 979.5164 111 980.4930 47 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_18 869 (77.5 108 978.5013 108 979.5030 69 980.5178 44 980.0389 14 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_17 876 (78.097) AM (Cen,3, 80.00 1.70e4 682.7003 16957 669.8290 2064 683.0331 10842 820.8694 8363 683.3691 5064 937.9755 6609 1023.5580 6337 1131.0293;807 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_11 871 (77.661) AM (Cen,3, 80.00 1.31e4 682.7000 13092 669.8295 1592 683.0336 8515 820.8680 7303 683.3688 3854 1023.5582 5424 1024.0614 3528 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_05 868 (77.381) AM (Cen,3, 80.00 9.87e3 820.8698 9871 682.7010 6299 682.3688 4364 683.0327 4405 754.3452 1567 821.3695 8992 821.8718 4503 1023.5580 3239 1131.0281 1063 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_05 8 173 978.4968 173 979.5081 111 980.4874;54 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_17 8 210 978.5016 210 979.5123 125 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_11 8 143 978.5013 143 980.0084 49 Figure 4. Spectrum 550 – 1200 amu Figure 5. Selected Precursor A B C A B C A B C A B C Waters ® Protein Expression System Informatics RMS error =4.64 ppm CV Area =14.2% A= Coefficient of Variation of Intensity lower [ ] B= log10 Intensity higher [ ] vs. log10 Intensity lower [ ] C= Intensity ratio histogram lower [ ] vs. higher [ ] Human Serum + 5 picomoles Waters Protein Expression System Standards Figure 6. 5 picomoles/100 femtomoles In Human Serum Figure 7. 5 picomoles/500 femtomoles In Human Serum Figure 8. 500 femtomoles/250 femtomoles In Human Serum Figure 9. 250 femtomoles/100 femtomoles In Human Serum Figure 2. BPI Chromatograms A B C Figure 3. SIC 978.xxxx D E F Human Serum + 1 picomole Standards G H I Human Serum + 0.5 picomoles Standards J K L Human Serum + 0.25 picomoles Standards M N O Human Serum + 0.1 picomoles Standards 600 800 1000 1200 m /z 0 100 % 080603_497HL_QuanExpSerial_07 869 (77.529) AM (Cen 2.22e4 790.9260 682.7010 669.8299 791.4247 820.8712 1045.9894 1046.4917 1046.9948 600 800 1000 1200 m /z 0 100 % 080603_497HL_QuanExpSerial_01 862 (76.861) AM (Cen 1.59e4 790.9269 682.7028 669.8300 791.4244 820.8719 1045.9910 1046.4923 1046.9946 600 800 1000 1200 m /z 0 100 % 080603_497HL_QuanExpSerial_13 868 (77.397) AM (Cen 2.34e4 790.9265 682.7001 791.4242 820.8721 821.3696 1045.9897 821.8726 1046.4911 1046.9940 977 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_07 869 (77.529) AM (Cen 3.72e3 978.9915 978.4916 977.5114 979.4922 979.9962 980.4995 977 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_01 862 (76.861) AM (Cen 2.90e3 978.9931 978.4935 977.5165 979.4935 979.9962 980.5021 977 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_13 868 (77.397) AM (Cen 4.02e3 978.9918 978.4919 977.4807 979.4923 979.9935 980.4962 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_03 868 (77.388) AM (Cen,4, 85.00, Ar,12000.0,7 1.16e4 820.8706 11559 682.7019 6038 682.3677 4011 683.0347 4309 821.3704 10147 821.8724 4900 937.4814 3465 847.8323 2010 1023.5581 3184 1131.0259 1036 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_09 869 (77.506) AM (Cen,4, 85.00, Ar,12000.0,7 1.89e4 682.7043 18903 682.3699 13883 669.8304 1592 683.0348 10713 820.8704 10078 683.3688 4385 937.9755 6199 821.8717 4237 1023.5587 5929 1024.0601 3862 1131.0311 605 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_15 873 (77.869) AM (Cen,4, 85.00, Ar,12000.0,7 1.33e4 820.8704 13251 682.7021 9377 682.3683 6462 628.2473 1074 683.0341 6139 821.3695 11558 821.8725 5308 937.9795 4841 847.8305 1770 1023.5575 3903 1131.0261 885 977 978 979 980 m/z 0 100 % 080603_497HL_QuanExpSerial_03 869 (7 727 978.4924 977.5117 977.9971 978.9933 979.4988 979.9883 977 978 979 980 m/z 0 100 % 080603_497HL_QuanExpSerial_09 869 (7 689 978.9965 978.4922 977.5118 978.0760 979.4936 979.9967 977 978 979 980 m/z 0 100 % 080603_497HL_QuanExpSerial_15 874 (7 851 978.9916 978.4910 977.5134 977.9865 979.4878 979.9998 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_04 870 (77.611) AM (Cen,4, 85.00, Ar,12000 2.92e4 682.7063 682.3712 669.8302 683.0350 1023.5591 820.8697 683.3691 683.7068 937.9747 821.8708 938.9804 1024.0618 1024.5626 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_10 867 (77.291) AM (Cen,4, 85.00, Ar,12000 1.42e4 682.7024 682.3688 669.8305 820.8701 683.0345 683.3688 683.7063 1023.5593 937.9771 1024.0610 1024.5647 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_16 878 (78.312) AM (Cen,4, 85.00, Ar,12000 3.81e4 682.7018 669.8304 683.0366 1023.5592 937.9731 683.3690 820.8691 938.9786 1024.0597 1024.5624 1131.0239 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_04 870 388 978.4943 978.9926 979.4992 979.9919 980.4755 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_16 878 339 978.4979 978.9942 979.4996 980.4976 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_10 867 286 978.5023 978.9941 979.4996 979.9974 980.9815 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_06 876 (78.0 132 978.4932 132 979.5203 96 979.0084 72 980.4995 50 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_12 862 (76.8 131 978.5094 131 979.5164 111 980.4930 47 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_18 869 (77.5 108 978.5013 108 979.5030 69 980.5178 44 980.0389 14 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_17 876 (78.097) AM (Cen,3, 80.00 1.70e4 682.7003 16957 669.8290 2064 683.0331 10842 820.8694 8363 683.3691 5064 937.9755 6609 1023.5580 6337 1131.0293;807 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_11 871 (77.661) AM (Cen,3, 80.00 1.31e4 682.7000 13092 669.8295 1592 683.0336 8515 820.8680 7303 683.3688 3854 1023.5582 5424 1024.0614 3528 600 700 800 900 1000 1100 m/z 0 100 % 080603_497HL_QuanExpSerial_05 868 (77.381) AM (Cen,3, 80.00 9.87e3 820.8698 9871 682.7010 6299 682.3688 4364 683.0327 4405 754.3452 1567 821.3695 8992 821.8718 4503 1023.5580 3239 1131.0281 1063 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_05 8 173 978.4968 173 979.5081 111 980.4874;54 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_17 8 210 978.5016 210 979.5123 125 978 979 980 981 m/z 0 100 % 080603_497HL_QuanExpSerial_11 8 143 978.5013 143 980.0084 49 Figure 4. Spectrum 550 – 1200 amu Figure 5. Selected Precursor A B C A B C A B C A B C Waters ® Protein Expression System Informatics RMS error =4.64 ppm CV Area =14.2% A= Coefficient of Variation of Intensity lower [ ] B= log10 Intensity higher [ ] vs. log10 Intensity lower [ ] C= Intensity ratio histogram lower [ ] vs. higher [ ] Introduction Sample Preparation Data Collection Results and Discussion • The protein digestion protocols employed in this study provide quantitatively reproducible results even on complex protein mix- tures as complex such as human serum. • The analytical protocols employed in this study are capable of re- producibly measuring the intensity of peptides in complex protein digest mixtures over three to four orders of magnitude. • Waters Protein Expression System Informatics is capable of ex- tracting peptide accurate mass, chromatographic retention time, and intensity information from complex protein digest mixtures in a quantitatively reproducible manner. • The analytical protocols employed in this study demonstrate that the combination of exact mass and chromatographic retention time can provide a very unique signature for each peptide con- tained in a complex protein digest mixture. • Waters Protein Expression System Informatics uses peptide exact mass and retention time signature information to match and quan- titatively compare the intensity of like identical peptides contained in protein digestion mixtures of a comparable control and experi- mental state. Table 2. Peptide Mass Fingerprint Analysis of Student t-Test Results 1. 16/63 matches (25%). RABIT Name: Glycogen phosphorylase, muscle form (Myophosphor Index: 463045 MW: 97290 Da pI: 6.8 Sequence 979.5407 979.5464 -5.8 835 843 1 (R)LPAPDEKIP (-) 1117.5616 1117.5642 -2.3 415 425 0 (R)VAAAFPGDVDR (L) 1177.5519 1177.5530 -0.90 471 479 0 (K)DFYELEPHK (F) 1262.5911 1262.5945 -2.7 774 783 0 (K)VFADYEEYVK (C) 1278.5392 1278.5391 0.074 726 735 0 (R)GYNAQEYYDR (I) 1386.7427 1386.7494 -4.8 415 427 1 (R)VAAAFPGDVDRLR (R) 1440.7295 1440.7375 -5.5 522 533 0 (K)LLSYVDDEAFIR (D) 1442.6935 1442.6956 -1.5 279 290 0 (R)VLYPNDNFFEGK (E) 1550.7635 1550.7702 -4.3 508 520 0 (R)IGEEYISDLDQLR (K) 1566.7963 1566.7916 3.0 257 270 0 (K)DFNVGGYIQAVLDR (N) 1580.8292 1580.8324 -2.0 741 754 0 (R)QIIEQLSSGFFSPK (Q) 1663.8410 1663.8300 6.6 2Met-ox 603 618 1 (R) TVMIGGKAAPGYHM 1840.9268 1840.9234 1.9 279 293 1 (R)VLYPNDNFFEGKELR (L) 1853.9958 1853.9948 0.56 492 507 0 (R)WLVLCNPGLAEIIAER (I) 1874.9087 1874.9071 0.87 372 387 0 (K)TCAYTNHTVLPEALER (W) 1889.9995 1890.0085 -4.8 623 640 0 (K)LITAIGDVVNHDPVVGDR (L 2. 10/63 matches (15%). gi|30794280|ref|NP_851335.1| albumin [Bos taurus] Index: 351651 MW: 69324 Da pI: 5.8 Sequence 1163.6200 1163.6312 -9.6 66 75 0 (K)LVNELTEFAK (T) 1291.5995 1291.6026 -2.4 300 309 0 (K)ECCDKPLLEK (S) 1479.7871 1479.7960 -6.0 421 433 0 (K)LGEYGFQNALIVR (Y) 1502.6156 1502.6143 0.85 375 386 0 (K)EYEATLEECCAK (D) 1554.6537 1554.6535 0.14 387 399 0 (K)DDPHACYSTVFDK (L) 1576.7693 1576.7681 0.75 139 151 0 (K)LKPDPNTLCDEFK (A) 3. 6/63 matches (9%). YEAST Name: Enolase 1 (2-phosphoglycerate dehydratase) (2-ph Index: 213440 MW: 46802 Da pI: 6.2 Sequence 807.4390 807.4365 3.1 179 185 0 (K)TFAEALR (I) 1159.6121 1159.6111 0.82 186 195 0 (R)IGSEVYHNLK (S) 1373.6436 1373.6411 1.8 244 255 0 (K)IGLDCASSEFFK (D) 1416.7140 1416.7222 -5.8 16 28 0 (R)GNPTVEVELTTEK (G) 1578.7960 1578.8015 -3.5 89 103 0 (K)AVDDFLISLDGTANK (S) 1840.9268 1840.9227 2.2 33 50 0 (R)SIVPSGASTGVHEALEMR (D) 4. 6/63 matches (9%). YEAST Name: Alcohol dehydrogenase I (YADH-1) Acc. #: 171025 Species: UNREADABLE Name: gi|171025|gb|AAA344 Index: 343993 MW: 36823 Da pI: 6.3 Sequence 1071.6357 1071.6414 -5.3 225 234 1 (K)EKDIVGAVLK (A) 1136.5694 1136.5740 -4.1 9 18 0 (K)GVIFYESHGK (L) 1355.6079 1355.6088 -0.65 277 287 0 (K)CCSDVFNQVVK (S) 1386.7427 1386.7415 0.86 262 276 0 (R)ANGTTVLVGMPAGAK (C) 1447.7940 1447.8048 -7.5 320 332 0 (K)VVGLSTLPEIYEK (M) 2312.1337 2312.1482 -6.3 235 258 0 (K)ATDGGAHGVINVSVSEAAIEA 35. 6/63 matches (11%). gi|27819608|ref|NP_776342.1| hemoglobin, beta [beta globin] Acc. #: 122572 Species: BOVIN Name: Hemoglobin beta chain Index: 521112 MW: 15955 Da pI: 7.0 Sequence 1328.7186 1328.7174 0.90 17 29 1 (K)VKVDEVGGEALGR (L) 1422.7242 1422.7269 -1.9 120 131 0 (K)EFTPVLQADFQK (V) 1448.6836 1448.6844 -0.54 82 94 0 (K)GTFAALSELHCDK (L) 2089.9682 2089.9541 6.8 40 58 0 (R)FFESFGDLSTADAVMNNPK (V) 1177.6812 1177.6822 1.21 3 15 0 (K)VVAGVANALAHR(S) 821.4074 821.4094 2.27 1 7 0 (-) MLTAEEK(T) Summary