asms2004 label free quantitation
DESCRIPTION
Demonstration of alternate scanning LCMS (Multiplex LCMS) for label-free quantitation of proteins and peptides in complex protein mixtures.TRANSCRIPT
©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. Geromanos1; Keith Richardson1; Phillip Young1; Richard Denny1; Marc Gorenstein1; Guo-Zhong Li1; Timothy Riley1; Jeffrey Silva1; Gregory J. Opiteck2; Ashok R. Dongre2; and Stanley A. Hefta2 1Waters Corporation, Milford, MA; 2Bristol-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 Exact Mass Retention-Time (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 [Glu1]-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 picomolesWaters Protein Expression System
Standards
Figure 6.5 picomoles/100 femtomolesIn Human Serum
Figure 7. 5 picomoles/500 femtomolesIn Human Serum
Figure 8. 500 femtomoles/250 femtomolesIn Human Serum
Figure 9. 250 femtomoles/100 femtomolesIn Human Serum
Figure 2. BPI Chromatograms
A
B
C
Figure 3. SIC
978.xxxx
D
E
F
Human Serum + 1 picomoleStandards
G
H
I
Human Serum + 0.5 picomolesStandards
J
K
L
Human Serum + 0.25 picomolesStandards
M
N
O
Human Serum + 0.1 picomolesStandards
6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0
1 0 0
%
0 8 0 6 0 3 _ 4 9 7 H L _ Q u a n E x p S e r ia l_ 0 7 8 6 9 (7 7 .5 2 9 ) A M (C e n2 .2 2 e 47 9 0 .9 2 6 0
6 8 2 .7 0 1 0
6 6 9 .8 2 9 9
7 9 1 .4 2 4 7
8 2 0 .8 7 1 2
1 0 4 5 .9 8 9 41 0 4 6 .4 9 1 71 0 4 6 .9 9 4 8
6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0
1 0 0
%
0 8 0 6 0 3 _ 4 9 7 H L _ Q u a n E x p S e r ia l_ 0 1 8 6 2 (7 6 .8 6 1 ) A M (C e n1 .5 9 e 47 9 0 .9 2 6 9
6 8 2 .7 0 2 8
6 6 9 .8 3 0 0
7 9 1 .4 2 4 4
8 2 0 .8 7 1 9
1 0 4 5 .9 9 1 01 0 4 6 .4 9 2 31 0 4 6 .9 9 4 6
6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0
1 0 0
%
0 8 0 6 0 3 _ 4 9 7 H L _ Q u a n E x p S e r ia l_ 1 3 8 6 8 (7 7 .3 9 7 ) A M (C e n2 .3 4 e 47 9 0 .9 2 6 5
6 8 2 .7 0 0 1
7 9 1 .4 2 4 2
8 2 0 .8 7 2 1
8 2 1 .3 6 9 61 0 4 5 .9 8 9 78 2 1 .8 7 2 6
1 0 4 6 .4 9 1 11 0 4 6 .9 9 4 0
977 978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_07 869 (77.529) AM (Cen3.72e3978.9915978.4916
977.5114
979.4922
979.9962980.4995
977 978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_01 862 (76.861) AM (Cen2.90e3978.9931978.4935
977.5165
979.4935
979.9962
980.5021
977 978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_13 868 (77.397) AM (Cen4.02e3978.9918
978.4919
977.4807
979.4923
979.9935
980.4962
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_03 868 (77.388) AM (Cen,4, 85.00, Ar,12000.0,71.16e4820.8706
11559
682.70196038
682.36774011
683.03474309
821.370410147
821.87244900 937.4814
3465847.83232010
1023.55813184
1131.02591036
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_09 869 (77.506) AM (Cen,4, 85.00, Ar,12000.0,71.89e4682.7043
18903
682.369913883
669.83041592
683.034810713 820.8704
10078683.3688
4385937.9755
6199821.8717
4237
1023.55875929
1024.06013862
1131.0311605
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_15 873 (77.869) AM (Cen,4, 85.00, Ar,12000.0,71.33e4820.8704
13251682.7021
9377
682.36836462
628.24731074
683.03416139
821.369511558
821.87255308
937.97954841
847.83051770
1023.55753903
1131.0261885
977 978 979 980m/z0
100
%
080603_497HL_QuanExpSerial_03 869 (7727978.4924
977.5117977.9971
978.9933
979.4988
979.9883
977 978 979 980m/z0
100
%
080603_497HL_QuanExpSerial_09 869 (7689978.9965978.4922
977.5118978.0760
979.4936
979.9967
977 978 979 980m/z0
100
%
080603_497HL_QuanExpSerial_15 874 (7851978.9916978.4910
977.5134977.9865
979.4878
979.9998
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_04 870 (77.611) AM (Cen,4, 85.00, Ar,120002.92e4682.7063
682.3712
669.8302
683.0350
1023.5591820.8697683.3691
683.7068
937.9747821.8708 938.9804
1024.06181024.5626
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_10 867 (77.291) AM (Cen,4, 85.00, Ar,120001.42e4682.7024
682.3688
669.8305
820.8701683.0345
683.3688
683.7063
1023.5593937.9771
1024.06101024.5647
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_16 878 (78.312) AM (Cen,4, 85.00, Ar,120003.81e4682.7018
669.8304
683.0366
1023.5592937.9731683.3690
820.8691 938.9786
1024.0597
1024.56241131.0239
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_04 870 388978.4943
978.9926
979.4992
979.9919
980.4755
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_16 878 339978.4979
978.9942
979.4996
980.4976
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_10 867 286978.5023
978.9941
979.4996
979.9974
980.9815
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_06 876 (78.0132978.4932
132 979.520396
979.008472 980.4995
50
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_12 862 (76.8131978.5094
131 979.5164111
980.493047
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_18 869 (77.5108978.5013
108 979.503069
980.517844
980.038914
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_17 876 (78.097) AM (Cen,3, 80.001.70e4682.7003
16957
669.82902064
683.033110842 820.8694
8363683.36915064
937.97556609
1023.55806337
1131.0293;807
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_11 871 (77.661) AM (Cen,3, 80.001.31e4682.7000
13092
669.82951592
683.03368515 820.8680
7303683.3688
38541023.5582
54241024.0614
3528
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_05 868 (77.381) AM (Cen,3, 80.009.87e3820.8698
9871682.70106299
682.36884364
683.03274405
754.34521567
821.36958992
821.87184503
1023.55803239 1131.0281
1063978 979 980 981
m/z0
100
%
080603_497HL_QuanExpSerial_05 8173978.4968
173
979.5081111
980.4874;54
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_17 8210978.5016
210
979.5123125
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_11 8143978.5013
143
980.008449
Figure 4. Spectrum 550 –
1200 amu
Figure 5. Selected Precursor
A
B
C
A
B
C
A
B
C
A
B
C
Waters
®Pro
tein Ex
pressio
n Sy
stem In
form
atics
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 picomolesWaters Protein Expression System
Standards
Figure 6.5 picomoles/100 femtomolesIn Human Serum
Figure 7. 5 picomoles/500 femtomolesIn Human Serum
Figure 8. 500 femtomoles/250 femtomolesIn Human Serum
Figure 9. 250 femtomoles/100 femtomolesIn Human Serum
Figure 2. BPI Chromatograms
A
B
C
Figure 3. SIC
978.xxxx
D
E
F
Human Serum + 1 picomoleStandards
G
H
I
Human Serum + 0.5 picomolesStandards
J
K
L
Human Serum + 0.25 picomolesStandards
M
N
O
Human Serum + 0.1 picomolesStandards
6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0
1 0 0
%
0 8 0 6 0 3 _ 4 9 7 H L _ Q u a n E x p S e r ia l_ 0 7 8 6 9 (7 7 .5 2 9 ) A M (C e n2 .2 2 e 47 9 0 .9 2 6 0
6 8 2 .7 0 1 0
6 6 9 .8 2 9 9
7 9 1 .4 2 4 7
8 2 0 .8 7 1 2
1 0 4 5 .9 8 9 41 0 4 6 .4 9 1 71 0 4 6 .9 9 4 8
6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0
1 0 0
%
0 8 0 6 0 3 _ 4 9 7 H L _ Q u a n E x p S e r ia l_ 0 1 8 6 2 (7 6 .8 6 1 ) A M (C e n1 .5 9 e 47 9 0 .9 2 6 9
6 8 2 .7 0 2 8
6 6 9 .8 3 0 0
7 9 1 .4 2 4 4
8 2 0 .8 7 1 9
1 0 4 5 .9 9 1 01 0 4 6 .4 9 2 31 0 4 6 .9 9 4 6
6 0 0 8 0 0 1 0 0 0 1 2 0 0m /z0
1 0 0
%
0 8 0 6 0 3 _ 4 9 7 H L _ Q u a n E x p S e r ia l_ 1 3 8 6 8 (7 7 .3 9 7 ) A M (C e n2 .3 4 e 47 9 0 .9 2 6 5
6 8 2 .7 0 0 1
7 9 1 .4 2 4 2
8 2 0 .8 7 2 1
8 2 1 .3 6 9 61 0 4 5 .9 8 9 78 2 1 .8 7 2 6
1 0 4 6 .4 9 1 11 0 4 6 .9 9 4 0
977 978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_07 869 (77.529) AM (Cen3.72e3978.9915978.4916
977.5114
979.4922
979.9962980.4995
977 978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_01 862 (76.861) AM (Cen2.90e3978.9931978.4935
977.5165
979.4935
979.9962
980.5021
977 978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_13 868 (77.397) AM (Cen4.02e3978.9918
978.4919
977.4807
979.4923
979.9935
980.4962
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_03 868 (77.388) AM (Cen,4, 85.00, Ar,12000.0,71.16e4820.8706
11559
682.70196038
682.36774011
683.03474309
821.370410147
821.87244900 937.4814
3465847.83232010
1023.55813184
1131.02591036
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_09 869 (77.506) AM (Cen,4, 85.00, Ar,12000.0,71.89e4682.7043
18903
682.369913883
669.83041592
683.034810713 820.8704
10078683.3688
4385937.9755
6199821.8717
4237
1023.55875929
1024.06013862
1131.0311605
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_15 873 (77.869) AM (Cen,4, 85.00, Ar,12000.0,71.33e4820.8704
13251682.7021
9377
682.36836462
628.24731074
683.03416139
821.369511558
821.87255308
937.97954841
847.83051770
1023.55753903
1131.0261885
977 978 979 980m/z0
100
%
080603_497HL_QuanExpSerial_03 869 (7727978.4924
977.5117977.9971
978.9933
979.4988
979.9883
977 978 979 980m/z0
100
%
080603_497HL_QuanExpSerial_09 869 (7689978.9965978.4922
977.5118978.0760
979.4936
979.9967
977 978 979 980m/z0
100
%
080603_497HL_QuanExpSerial_15 874 (7851978.9916978.4910
977.5134977.9865
979.4878
979.9998
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_04 870 (77.611) AM (Cen,4, 85.00, Ar,120002.92e4682.7063
682.3712
669.8302
683.0350
1023.5591820.8697683.3691
683.7068
937.9747821.8708 938.9804
1024.06181024.5626
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_10 867 (77.291) AM (Cen,4, 85.00, Ar,120001.42e4682.7024
682.3688
669.8305
820.8701683.0345
683.3688
683.7063
1023.5593937.9771
1024.06101024.5647
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_16 878 (78.312) AM (Cen,4, 85.00, Ar,120003.81e4682.7018
669.8304
683.0366
1023.5592937.9731683.3690
820.8691 938.9786
1024.0597
1024.56241131.0239
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_04 870 388978.4943
978.9926
979.4992
979.9919
980.4755
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_16 878 339978.4979
978.9942
979.4996
980.4976
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_10 867 286978.5023
978.9941
979.4996
979.9974
980.9815
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_06 876 (78.0132978.4932
132 979.520396
979.008472 980.4995
50
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_12 862 (76.8131978.5094
131 979.5164111
980.493047
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_18 869 (77.5108978.5013
108 979.503069
980.517844
980.038914
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_17 876 (78.097) AM (Cen,3, 80.001.70e4682.7003
16957
669.82902064
683.033110842 820.8694
8363683.36915064
937.97556609
1023.55806337
1131.0293;807
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_11 871 (77.661) AM (Cen,3, 80.001.31e4682.7000
13092
669.82951592
683.03368515 820.8680
7303683.3688
38541023.5582
54241024.0614
3528
600 700 800 900 1000 1100m/z0
100
%
080603_497HL_QuanExpSerial_05 868 (77.381) AM (Cen,3, 80.009.87e3820.8698
9871682.70106299
682.36884364
683.03274405
754.34521567
821.36958992
821.87184503
1023.55803239 1131.0281
1063978 979 980 981
m/z0
100
%
080603_497HL_QuanExpSerial_05 8173978.4968
173
979.5081111
980.4874;54
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_17 8210978.5016
210
979.5123125
978 979 980 981m/z0
100
%
080603_497HL_QuanExpSerial_11 8143978.5013
143
980.008449
Figure 4. Spectrum 550 –
1200 amu
Figure 5. Selected Precursor
A
B
C
A
B
C
A
B
C
A
B
C
Waters
®Pro
tein Ex
pressio
n Sy
stem In
form
atics
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