Siena Simultaneous Qualitative and Quantitative Analysis of ......MPDS. Exact mass LC/MS data was...
1
©2004 Waters Corporation 720001013 Simultaneous Qualitative and Quantitative Analysis of Whole Proteomes and Complex Protein Mixtures using Accurate Mass and Chromatographic Information Roy Martin 1 , Jeff Silva 2 , Keith Richardson 3 , Kieran Neeson 3 , Scott Geromanos 2 (Waters Corp. 1 Beverly, MA; 2 Milford, MA, USA. 3 Manchester, UK); Siena 2004 This poster illustrates the quantitative and qualitative capabilities of the new Waters ® Protein Expression System. This system was designed to analyze complex mixtures and quantify changes in protein levels through the relative area difference of accurately measured peptide chromatographic peaks. Since data were acquired in triplicate in alternate scanning low and elevated energy accurate mass LC/MS mode, each peptide measurement has both quantita- tive statistics and fragmentation information for qualitative assessment. The alternate scan method has considerably higher duty cycle than traditional data-directed analyses which are performed in a serial manner. It also makes full use of the speed and accuracy of an OA-Tof analyzer. Data presented are from two sources: E. Coli cells grown under different carbon source con- ditions, and commercially available tryptically digested human serum samples spiked with dif- ferent concentrations of five exogenous internal reference proteins contained in the Waters Protein Expression System Standards. Raw data was 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 processed further to match and compare the intensity of like EMRT components from different sample injections. The accurate masses are then matched with available fragment ions for qualitative assessment. Precursor ions and frag- ment ions are related by chromatographic peak maxima that they share. Once peptide iden- tities are assigned, the data can be further processed to yield quantitative results based on protein identities. Introduction Sample Preparation Results and Discussion E. Coli Data The protein digestion protocols employed in this study provide quantitatively re- producible results even on complex protein mixtures as complex as human se- rum. The analytical protocols employed in this study are capable of reproducibly meas- uring the intensity of peptides in complex protein digest mixtures over three to four orders of magnitude. Waters Protein Expression System Informatics is capable of extracting 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 contained in a complex protein digest mixture. Waters Protein Expression System Informatics uses peptide exact mass and re- tention time signature information to match and quantitatively compare the in- tensity of identical peptides contained in protein digestion mixtures of a compa- rable control and experimental state. Comprehensive data sets are generated which can be extensively mined for fur- ther qualitative and quantitative information. Summary Data Collection Figure 1. Data acquisition using the Waters Protein Expression System mass spectrometer (Q-Tof). The instrument acquires data in alternate high and low collision energy scans to generate a compre- hensive data where one set of scans contains mostly precursor ions, and a simultaneous set of scans contains mainly fragment ions. Human serum (Sigma source) was tryptically digested according to the procedure (C. Dorschel et al., “Protocols to Assure Reproducible Quantitative and Qualitative Analysis of Tryptic Digests of Complex Pro- tein Mixtures for Global Proteomic Experiments”). Five aliquots of this human serum digest were spiked with Waters Mass Prep Digest Standards (MPDS), containing equimolar levels of Yeast Enolase and Alcohol De- hydrogenase, Rabbit Glycogen Phosphorylate, Bovine Serum Albumin and Hemoglobin tryptic digest. E. Coli Media and Growth Conditions: Frozen E. coli (ATCC10798, K-12) cell stocks were streaked onto Luria-Bertani (LB) plates and grown at 37 degrees Celsius. An individual colony was subsequently streaked onto M9 minimal medium plated supplemented with 0.5% sodium acetate and grown at 37 degrees Celsius. Seed cultures were generated by transferring single colonies into flasks of M9 minimal media supplemented with 0.5% sodium acetate. Seed culture flasks were shaken at 250 rpm at 37 degrees Celsius until mid log phase (OD600 = 0.9-1.1). The seed culture was diluted 1ml:500ml into separate M9 minimal media supple- mented with one of three carbon sources (0.5% glucose, 0.5% lactose or 0.5% sodium acetate). Flasks were shaken at 250 rpm at 37 degrees Celsius until mid log phase (OD600 = 0.9-1.1). The E. coli cell cul- tures were harvested by centrifugation, pellets were frozen at -80 degrees Celsius. E. Coli Protein Extract Preparation: Frozen cells were suspended in 5ml per 1gm biomass in lysis buffer (Dulbecco’s Phosphate-Buffered Saline + 1/100 Protease Inhibitor Cocktail [Sigma cat #8340]) in a 50 mL Falcon tube. The cells were lysed by sonication in a Microson XL Ultrasonic Cell Disrupter (Misonix, Inc.) at 4 degrees Celsius. The cell debris was removed by centrifugation at 15,000xg for 30minutes at 4c, and the resulting soluble protein extracts were dispensed into 1 mL aliquots and stored at -80 degree Celsius for sub- sequent analysis. LC/MS System: Waters Protein Expression System comprised of the Waters CapLC ® System with the Wa- ters Micromass ® Q-Tof Ultima API Mass Spectrometer equipped with a NanoLockSpray™ Source oper- ated 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 E. Coli extract digests and five spiked human serum (HS) digest samples was analyzed in tripli- cate. Injections were 5 microliters each and were made directly on-column. The total protein load for each injection was ~8.5 micrograms of Human Serum plus either 5.0, 1.0, 0.5, 0.25 or 0.1 picomoles of the spiked MPDS. Exact mass LC/MS data was collected 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 ac- quired every 4.0 seconds. The NanoLockSpray source was switched every 10 seconds to obtain a reference scan of [Glu 1 ]-Fibrinopeptide lockmass calibrant. Compare measured high energy fragment ions to theoretical y and b ions Assign sequence and match to protein Normalize intensity to endogenous internal standard peptide(s) qualitative Compare measured high energy fragment ions to theoretical y and b ions Assign sequence and match to protein Normalize intensity to endogenous internal standard peptide(s) match/compare match/compare quantitative LC-MS Accurate Mass Analysis in Alternating High and Low Collision Energy Mode Extract exact mass, retention time, and intensity for the monoisotopic ion For every unique peptide in mixture Compare measured low energy peptide exact masses to theoretical masses Control State Complex Protein Mixture Tryptic Digest Compare measured high energy fragment ions to theoretical y and b ions Assign sequence and match to protein Normalize intensity to endogenous internal standard peptide(s) qualitative LC-MS Accurate Mass Analysis in Alternating High and Low Collision Energy Mode Extract exact mass, retention time, and intensity for the monoisotopic ion for every unique peptide in mixture Compare measured low energy peptide exact masses to theoretical masses Experimental State Complex Protein Mixture Tryptic Digest Compare measured high energy fragment ions to theoretical y and b ions Assign sequence and match to protein Normalize intensity to endogenous internal standard peptide(s) match/compare match/compare quantitative Accession #7 + Accession #6 ++++++ Accession #5 ++ Accession #4 ++ Accession #3 ++ Accession #2 ++++++ Accession #1 + Protein Assignment Peptide Score Candidate Peptide Sequence Candidate Peptide Mass Accession #7 + Accession #6 ++++++ Accession #5 ++ Accession #4 ++ Accession #3 ++ Accession #2 ++++++ Accession #1 + Protein Assignment Peptide Score Candidate Peptide Sequence Candidate Peptide Mass PLG 2.0 Protein Sequence dB (peptide fragments) MS E y”1 y” n bn b1 y”1 y” n bn b1 EE Figure 7 The spectra below show the effect of the cleaning process. The top panel shows the deconvoluted masses that apex at 54.81 minutes in Figure 4. The second and third panels show matches to two different peptides. S1 Acetate m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 % 0 100 040514_497_V_EcoliBS_09_Ac 680 (54.844) 2: TOF MS ES+ 296 171.1523 136.1090 129.1333 227.2341 298.2824 229.1753 906.6710 399.3235 300.2301 312.2588 313.2615 905.6271 620.4705 461.3834 600.4336 462.3390 848.6085 621.4566 724.5336 699.5518 767.6749 1006.7227 941.6794 1216.9331 1007.7325 1008.7688 1173.8735 1217.9011 1217.9723 1218.9120 1268.8640 1332.9530 Figure 6 Protein Expression System Informatics uses this principle of chro- matographic apex alignment as a first filter to remove ions from the elevated energy spectra that may have a retention time similar to, but not coincident with a parent ion of interest . This spectrum shows the combined elevated energy spectra of all ions that elute at 54.81 minutes. The result of this cleaning process is illustrated in Figure 7. T im e 20.00 40.00 60.00 80.00 100.00 % 0 100 040514_497_V_EcoliBS_09_Ac 1: TOF MS ES+ BPI 3.82e3 17.07 15.64 33.42 28.12 17.74 19.62 21.19 39.44 45.76 65.03 46.06 53.29 49.84 59.19 70.22 71.34 85.01 72.09 80.96 77.73 102.74 95.11 85.31 89.66 104.32 116.41 Scan 54.809 minute S1 Acetate T im e 20.00 40.00 60.00 80.00 100.00 % 0 100 040514_497_V_EcoliBS_09_Ac 1: TOF MS ES+ BPI 3.82e3 17.07 15.64 33.42 28.12 17.74 19.62 21.19 39.44 45.76 65.03 46.06 53.29 49.84 59.19 70.22 71.34 85.01 72.09 80.96 77.73 102.74 95.11 85.31 89.66 104.32 116.41 Scan 54.809 minute Cleaned High-Energy Data RT Apex = 54.81 Succinyl CoA Synthase ß chain VALDPLTGPMPYQGR MH+ 1614.8426 Isocitrate Lyase LLAYNBSPSFNWQK MH+ 1727.8324 Figure 8 Candidate Peptides are identified by matching accurately measured masses against a database con- taining all possible tryptic frag- ments with no or one missed cleavages. This prescreen gener- ates a smaller table of candidate peptide sequences. For positive identification, the ions observed in the co-apexing cleaned high en- ergy data are compared against a model fragmentation pattern gen- erated from the database peptide sequence. At the mass accura- cies typically used (<5ppm) the correct sequence is usually clearly distinguished from incorrect se- quences. Low-Energy Elevated-Energy Elevated-Energy 1 Low-Energy Elevated-Energy Elevated-Energy Low-Energy Elevated-Energy Elevated-Energy 1 Intensity Plot E. Coli grown on Acetate Injection 1 Vs Injection 2 Spiked Serum Data Figure 2. Flowchart for Quantitative and Qualititative analysis. Two sample states are shown, although the method is not limited and the software can handle up to 300 comparisons. Figure 3. Base peak intensity plot of E. Coli digest from cell extract grown on minimal acetate as a carbon source Figure 4. This shows that while a large number of ions may be present in a single spectrum, the software distinguished between ions that have different peak maxima. In this example, two red ions share the exact same retention time, as do their related fragment ions. Figure 10 The three panels above illustrate the result of comparing E. Coli grown on glucose with those grown on Acetate. Data points indicate log intensity of compared peptides in each sample. Panels A and B show how different injections of the same lysate digest compare with each other. The straight diagonal line shows little variation in intensity between replicate injections. Panel C is the result when cells grown in acetate are compared with those grown in glucose. The wide dispersion of peptide intensities, as would be expected when E. Coli are grown under conditions that involve a different set of metabolic pathways. Figure 9 Peptides are clustered based on mass and retention time pairs. Quantification is based on accurately measure chromatographic areas of the deconvoluted molecular mass. The example at left shows the response curves for 6 of the EMRT’ associated with the MPDS over five spiked levels. Those EMRT’s associated with serum had a slope of zero. Peptides can be quantified on their own, or as sets of peptides identified in da- tabases then quantified using protein ID’s as a basis for comparison. m/z 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 % 0 100 040514_497_V_EcoliBS_09_Ac 680 (54.809) 1: TOF MS ES+ 1.20e3 808.1044 643.4724 610.4412 597.7974 510.3848 525.3868 643.8036 644.1348 644.4870 716.0485 676.4930 777.5513 765.0887 808.5915 865.1115 809.1020 809.6127 849.6427 865.6155 870.6758 871.1934 922.1773 871.6631 896.1744 941.6919 950.7143 965.2219 965.7289 1062.7375 1143.8635 m/z 609 610 611 612 613 614 615 616 617 618 % 0 100 040514_497_V_EcoliBS_09_Ac 680 (54.809) 1: TOF MS ES+ 250 610.4412 608.4672 610.4613 610.9452 611.9439 612.4486 612.9332 615.4911 614.7728 617.9631 S1 Acetate m/z 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 % 0 100 040514_497_V_EcoliBS_09_Ac 680 (54.809) 1: TOF MS ES+ 1.20e3 808.1044 643.4724 610.4412 597.7974 510.3848 525.3868 643.8036 644.1348 644.4870 716.0485 676.4930 777.5513 765.0887 808.5915 865.1115 809.1020 809.6127 849.6427 865.6155 870.6758 871.1934 922.1773 871.6631 896.1744 941.6919 950.7143 965.2219 965.7289 1062.7375 1143.8635 S1 Acetate m/z 609 610 611 612 613 614 615 616 617 618 % 0 100 040514_497_V_EcoliBS_09_Ac 680 (54.809) 1: TOF MS ES+ 250 610.4412 608.4672 610.4613 610.9452 611.9439 612.4486 612.9332 615.4911 614.7728 617.9631 Figure 5. Single MS scan at 54.8 minutes from E. Coli sample. Insert shows resolution and peak shape, and illus- trates an example where a low resolution scan would have difficulty picking the correct precursor mass, and a DDA analysis would most likely pass both precursor ions, confusing the MS/MS interpretation.. Quantitative Principle Qualitative Principle Intensity Plot E. Coli grown on Acetate Injection 1 Vs Injection 2 A B C Figure 11 This plot is the comparison of two of the different spiked serum samples. Serum spiked with 2.5 pmol of pro- tein standard is plotted on the vertical axis, and serum spiked with 0.5 pmol protein standard is spiked on the horizontal axis. The circled points off the diagonal axis correspond to the peptides from the differentially spiked proteins. The accurate masses of these peptides can be used to identify the proteins alone, or they can be combined with the high energy data for further confirmation as shown below. Figure 12 The quantitative viewer (left) and the qualitative viewer (below) are integrated into existing software. Relative quantification uses protein ID’s, and the results are indi- cated by the initial number in the columns at left. The standard deviation follows the relative quan level, and the P-value for the expression is in parentheses. A P-value approaches 1 for a definite up-regulation, and approaches 0 for a definite down-regulation. The 95% confidence lev- els are between 0.05 and .95. Internal standards are high- lighted in yellow; they can be endogenous, spiked or both. Log intensity of 2.5pmol vs. 0.5 pmol Intensity Plot E. Coli grown on Glucose Injection 1 Vs Injection 2 Concepts of Data Processing Intensity Plot E. Coli grown on Glucose vs. E. Coli grown on Acetate
Transcript of Siena Simultaneous Qualitative and Quantitative Analysis of ......MPDS. Exact mass LC/MS data was...
©2004 Waters Corporation 720001013
Simultaneous Qualitative and Quantitative Analysis of Whole Proteomes and Complex Protein Mixtures using Accurate Mass and Chromatographic Information
Roy Martin1, Jeff Silva2, Keith Richardson3, Kieran Neeson3, Scott Geromanos2 (Waters Corp.1Beverly, MA; 2Milford, MA, USA. 3Manchester, UK);
Siena 2004
This poster illustrates the quantitative and qualitative capabilities of the new Waters® Protein Expression System. This system was designed to analyze complex mixtures and quantify changes in protein levels through the relative area difference of accurately measured peptide chromatographic peaks. Since data were acquired in triplicate in alternate scanning low and elevated energy accurate mass LC/MS mode, each peptide measurement has both quantita-tive statistics and fragmentation information for qualitative assessment. The alternate scan method has considerably higher duty cycle than traditional data-directed analyses which are performed in a serial manner. It also makes full use of the speed and accuracy of an OA-Tof analyzer. Data presented are from two sources: E. Coli cells grown under different carbon source con-ditions, and commercially available tryptically digested human serum samples spiked with dif-ferent concentrations of five exogenous internal reference proteins contained in the Waters Protein Expression System Standards. Raw data was 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 processed further to match and compare the intensity of like EMRT components from different sample injections. The accurate masses are then matched with available fragment ions for qualitative assessment. Precursor ions and frag-ment ions are related by chromatographic peak maxima that they share. Once peptide iden-tities are assigned, the data can be further processed to yield quantitative results based on protein identities.
Introduction
Sample Preparation
Results and Discussion E. Coli Data
The protein digestion protocols employed in this study provide quantitatively re-producible results even on complex protein mixtures as complex as human se-rum.
The analytical protocols employed in this study are capable of reproducibly meas-uring the intensity of peptides in complex protein digest mixtures over three to four orders of magnitude.
Waters Protein Expression System Informatics is capable of extracting 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 contained in a complex protein digest mixture.
Waters Protein Expression System Informatics uses peptide exact mass and re-tention time signature information to match and quantitatively compare the in-tensity of identical peptides contained in protein digestion mixtures of a compa-rable control and experimental state.
Comprehensive data sets are generated which can be extensively mined for fur-ther qualitative and quantitative information.
Summary
Data Collection
Figure 1. Data acquisition using the Waters Protein Expression System mass spectrometer (Q-Tof). The instrument acquires data in alternate high and low collision energy scans to generate a compre-hensive data where one set of scans contains mostly precursor ions, and a simultaneous set of scans contains mainly fragment ions.
Human serum (Sigma source) was tryptically digested according to the procedure (C. Dorschel et al., “Protocols to Assure Reproducible Quantitative and Qualitative Analysis of Tryptic Digests of Complex Pro-tein Mixtures for Global Proteomic Experiments”). Five aliquots of this human serum digest were spiked with Waters Mass Prep Digest Standards (MPDS), containing equimolar levels of Yeast Enolase and Alcohol De-hydrogenase, Rabbit Glycogen Phosphorylate, Bovine Serum Albumin and Hemoglobin tryptic digest. E. Coli Media and Growth Conditions: Frozen E. coli (ATCC10798, K-12) cell stocks were streaked onto Luria-Bertani (LB) plates and grown at 37 degrees Celsius. An individual colony was subsequently streaked onto M9 minimal medium plated supplemented with 0.5% sodium acetate and grown at 37 degrees Celsius. Seed cultures were generated by transferring single colonies into flasks of M9 minimal media supplemented with 0.5% sodium acetate. Seed culture flasks were shaken at 250 rpm at 37 degrees Celsius until mid log phase (OD600 = 0.9-1.1). The seed culture was diluted 1ml:500ml into separate M9 minimal media supple-mented with one of three carbon sources (0.5% glucose, 0.5% lactose or 0.5% sodium acetate). Flasks were shaken at 250 rpm at 37 degrees Celsius until mid log phase (OD600 = 0.9-1.1). The E. coli cell cul-tures were harvested by centrifugation, pellets were frozen at -80 degrees Celsius. E. Coli Protein Extract Preparation: Frozen cells were suspended in 5ml per 1gm biomass in lysis buffer (Dulbecco’s Phosphate-Buffered Saline + 1/100 Protease Inhibitor Cocktail [Sigma cat #8340]) in a 50 mL Falcon tube. The cells were lysed by sonication in a Microson XL Ultrasonic Cell Disrupter (Misonix, Inc.) at 4 degrees Celsius. The cell debris was removed by centrifugation at 15,000xg for 30minutes at 4c, and the resulting soluble protein extracts were dispensed into 1 mL aliquots and stored at -80 degree Celsius for sub-sequent analysis. LC/MS System: Waters Protein Expression System comprised of the Waters CapLC® System with the Wa-
ters Micromass® Q-Tof Ultima API Mass Spectrometer equipped with a NanoLockSpray™ Source oper-ated 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 E. Coli extract digests and five spiked human serum (HS) digest samples was analyzed in tripli-cate. Injections were 5 microliters each and were made directly on-column. The total protein load for each injection was ~8.5 micrograms of Human Serum plus either 5.0, 1.0, 0.5, 0.25 or 0.1 picomoles of the spiked MPDS. Exact mass LC/MS data was collected 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 ac-quired every 4.0 seconds. The NanoLockSpray source was switched every 10 seconds to obtain a reference scan of [Glu1]-Fibrinopeptide lockmass calibrant.
LC-MS Accurate Mass Analysis in Alternating High and Low Collision
Energy Mode
Extract exact mass, retention time, and intensity for the monoisotopic ion
For every unique peptide in mixture
Compare measured low energy peptide exact masses to theoretical masses
Control StateComplex Protein Mixture Tryptic Digest
Compare measured high energy fragmentions to theoretical y and b ions
Assign sequence and match to protein
Normalize intensity to endogenousinternal standard peptide(s)
qualitative
LC-MS Accurate Mass Analysis in Alternating High and Low Collision
Energy Mode
Extract exact mass, retention time, and intensity for the monoisotopic ion
for every unique peptide in mixture
Compare measured low energy peptide exact masses to theoretical masses
Experimental StateComplex Protein Mixture Tryptic Digest
Compare measured high energy fragmentions to theoretical y and b ions
Assign sequence and match to protein
Normalize intensity to endogenousinternal standard peptide(s)
match/compare
match/comparequantitative
LC-MS Accurate Mass Analysis in Alternating High and Low Collision
Energy Mode
Extract exact mass, retention time, and intensity for the monoisotopic ion
For every unique peptide in mixture
Compare measured low energy peptide exact masses to theoretical masses
Control StateComplex Protein Mixture Tryptic Digest
Compare measured high energy fragmentions to theoretical y and b ions
Assign sequence and match to protein
Normalize intensity to endogenousinternal standard peptide(s)
qualitative
LC-MS Accurate Mass Analysis in Alternating High and Low Collision
Energy Mode
Extract exact mass, retention time, and intensity for the monoisotopic ion
for every unique peptide in mixture
Compare measured low energy peptide exact masses to theoretical masses
Experimental StateComplex Protein Mixture Tryptic Digest
Compare measured high energy fragmentions to theoretical y and b ions
Assign sequence and match to protein
Normalize intensity to endogenousinternal standard peptide(s)
match/compare
match/comparequantitative
Accession #7+
Accession #6++++++
Accession #5++
Accession #4++
Accession #3++
Accession #2++++++
Accession #1+
ProteinAssignment
PeptideScore
Candidate Peptide SequenceCandidate Peptide Mass
Accession #7+
Accession #6++++++
Accession #5++
Accession #4++
Accession #3++
Accession #2++++++
Accession #1+
ProteinAssignment
PeptideScore
Candidate Peptide SequenceCandidate Peptide Mass
PLG 2.0Protein
Sequence dB (peptide fragments)
MSE
y”1y”nbnb1y”1y”nbnb1
EE
Figure 7 The spectra below show the effect of the cleaning process. The top panel shows the deconvoluted masses that apex at 54.81 minutes in Figure 4. The second and third panels show matches to two different peptides.
S1 Acetate
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0
100040514_497_V_EcoliBS_09_Ac 680 (54.844) 2: TOF MS ES+
296171.1523
136.1090
129.1333
227.2341
298.2824
229.1753 906.6710399.3235
300.2301
312.2588
313.2615
905.6271620.4705
461.3834 600.4336462.3390
848.6085
621.4566
724.5336699.5518 767.6749
1006.7227
941.67941216.9331
1007.7325
1008.76881173.8735
1217.9011
1217.9723
1218.9120
1268.86401332.9530
Figure 6 Protein Expression System Informatics uses this principle of chro-matographic apex alignment as a first filter to remove ions from the elevated energy spectra that may have a retention time similar to, but not coincident with a parent ion of interest . This spectrum shows the combined elevated energy spectra of all ions that elute at 54.81 minutes. The result of this cleaning process is illustrated in Figure 7.
S 1 A c e t a t e
T im e2 0 .0 0 4 0 .0 0 6 0 .0 0 8 0 .0 0 1 0 0 .0 0
%
0
1 0 00 4 0 5 1 4 _ 4 9 7 _ V _ E c o liB S _ 0 9 _ A c 1 : T O F M S E S +
B P I3 .8 2 e 3
1 7 .0 7
1 5 .6 4
3 3 .4 2
2 8 .1 2
1 7 .7 4
1 9 .6 2
2 1 .1 9
3 9 .4 4 4 5 .7 6 6 5 .0 3
4 6 .0 6 5 3 .2 94 9 .8 4
5 9 .1 9
7 0 .2 2
7 1 .3 48 5 .0 1
7 2 .0 9
8 0 .9 67 7 .7 3
1 0 2 .7 49 5 .1 1
8 5 .3 1
8 9 .6 6
1 0 4 .3 2
1 1 6 .4 1
Scan 54.809 m inute
S 1 A c e t a t e
T im e2 0 .0 0 4 0 .0 0 6 0 .0 0 8 0 .0 0 1 0 0 .0 0
%
0
1 0 00 4 0 5 1 4 _ 4 9 7 _ V _ E c o liB S _ 0 9 _ A c 1 : T O F M S E S +
B P I3 .8 2 e 3
1 7 .0 7
1 5 .6 4
3 3 .4 2
2 8 .1 2
1 7 .7 4
1 9 .6 2
2 1 .1 9
3 9 .4 4 4 5 .7 6 6 5 .0 3
4 6 .0 6 5 3 .2 94 9 .8 4
5 9 .1 9
7 0 .2 2
7 1 .3 48 5 .0 1
7 2 .0 9
8 0 .9 67 7 .7 3
1 0 2 .7 49 5 .1 1
8 5 .3 1
8 9 .6 6
1 0 4 .3 2
1 1 6 .4 1
Scan 54.809 m inute
Cleaned High-Energy Data
RT Apex = 54.81
Succinyl CoA Synthase ß chain VALDPLTGPMPYQGR
MH+ 1614.8426
Isocitrate Lyase LLAYNBSPSFNWQK
MH+ 1727.8324
Figure 8 Candidate Peptides are identified by matching accurately measured masses against a database con-taining all possible tryptic frag-ments with no or one missed cleavages. This prescreen gener-ates a smaller table of candidate peptide sequences. For positive identification, the ions observed in the co-apexing cleaned high en-ergy data are compared against a model fragmentation pattern gen-erated from the database peptide sequence. At the mass accura-cies typically used (<5ppm) the correct sequence is usually clearly distinguished from incorrect se-quences.
Low-EnergyElevated-Energy Elevated-Energy
1
Low-EnergyElevated-Energy Elevated-EnergyLow-EnergyElevated-Energy Elevated-Energy
1
Intensity Plot E. Coli grown on Acetate Injection 1 Vs Injection 2
Spiked Serum Data
Figure 2. Flowchart for Quantitative and Qualititative analysis. Two sample states are shown, although the method is not limited and the software can handle up to 300 comparisons.
Figure 3. Base peak intensity plot of E. Coli digest from cell extract grown on minimal acetate as a carbon source
Figure 4. This shows that while a large number of ions may be present in a single spectrum, the software distinguished between ions that have different peak maxima. In this example, two red ions share the exact same retention time, as do their related fragment ions.
Figure 10 The three panels above illustrate the result of comparing E. Coli grown on glucose with those grown on Acetate. Data points indicate log intensity of compared peptides in each sample. Panels A and B show how different injections of the same lysate digest compare with each other. The straight diagonal line shows little variation in intensity between replicate injections. Panel C is the result when cells grown in acetate are compared with those grown in glucose. The wide dispersion of peptide intensities, as would be expected when E. Coli are grown under conditions that involve a different set of metabolic pathways.
Figure 9 Peptides are clustered based on mass and retention time pairs. Quantification is based on accurately measure chromatographic areas of the deconvoluted molecular mass. The example at left shows the response curves for 6 of the EMRT’ associated with the MPDS over five spiked levels. Those EMRT’s associated with serum had a slope of zero. Peptides can be quantified on their own, or as sets of peptides identified in da-tabases then quantified using protein ID’s as a basis for comparison.
S1 Acetate
m /z450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300
%
0
100040514_497_V_EcoliBS_09_Ac 680 (54.809) 1: TOF MS ES+
1.20e3808.1044
643.4724
610.4412
597.7974
510.3848525.3868
643.8036
644.1348
644.4870716.0485
676.4930 777.5513765.0887
808.5915
865.1115
809.1020
809.6127
849.6427
865.6155
870.6758
871.1934
922.1773
871.6631
896.1744
941.6919
950.7143
965.2219
965.72891062.7375 1143.8635
S1 Acetate
m /z609 610 611 612 613 614 615 616 617 618
%
0
100040514_497_V_EcoliBS_09_Ac 680 (54.809) 1: TOF MS ES+
250610.4412
608.4672
610.4613
610.9452
611.9439
612.4486
612.9332
615.4911614.7728 617.9631
S1 Acetate
m /z450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300
%
0
100040514_497_V_EcoliBS_09_Ac 680 (54.809) 1: TOF MS ES+
1.20e3808.1044
643.4724
610.4412
597.7974
510.3848525.3868
643.8036
644.1348
644.4870716.0485
676.4930 777.5513765.0887
808.5915
865.1115
809.1020
809.6127
849.6427
865.6155
870.6758
871.1934
922.1773
871.6631
896.1744
941.6919
950.7143
965.2219
965.72891062.7375 1143.8635
S1 Acetate
m /z609 610 611 612 613 614 615 616 617 618
%
0
100040514_497_V_EcoliBS_09_Ac 680 (54.809) 1: TOF MS ES+
250610.4412
608.4672
610.4613
610.9452
611.9439
612.4486
612.9332
615.4911614.7728 617.9631
Figure 5. Single MS scan at 54.8 minutes from E. Coli sample. Insert shows resolution and peak shape, and illus-trates an example where a low resolution scan would have difficulty picking the correct precursor mass, and a DDA analysis would most likely pass both precursor ions, confusing the MS/MS interpretation..
Quantitative Principle
Qualitative Principle
Intensity Plot E. Coli grown on Acetate Injection 1 Vs Injection 2
A B
C
Figure 11 This plot is the comparison of two of the different spiked serum samples. Serum spiked with 2.5 pmol of pro-tein standard is plotted on the vertical axis, and serum spiked with 0.5 pmol protein standard is spiked on the horizontal axis. The circled points off the diagonal axis correspond to the peptides from the differentially spiked proteins. The accurate masses of these peptides can be used to identify the proteins alone, or they can be combined with the high energy data for further confirmation as shown below.
Figure 12 The quantitative viewer (left) and the qualitative viewer (below) are integrated into existing software. Relative quantification uses protein ID’s, and the results are indi-cated by the initial number in the columns at left. The standard deviation follows the relative quan level, and the P-value for the expression is in parentheses. A P-value approaches 1 for a definite up-regulation, and approaches 0 for a definite down-regulation. The 95% confidence lev-els are between 0.05 and .95. Internal standards are high-lighted in yellow; they can be endogenous, spiked or both.
Log intensity of 2.5pmol vs. 0.5 pmol
Intensity Plot E. Coli grown on Glucose Injection 1 Vs Injection 2
Concepts of Data Processing
Intensity Plot E. Coli grown on Glucose
vs. E. Coli grown on Acetate
