Asms2004 Alternate Scanning Lcms

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Demonstration of alternate scanning LCMS for simultanous acquisition of precursor and product ions without precursor mass selection (ie Multiplex LCMS).

Transcript of Asms2004 Alternate Scanning Lcms

  • 2004 Waters Corporation

    Traditional tandem mass spectrometry (MS/MS) is a method of mass spectrometry whereby intact peptides (parent ions) generated from an enzymatic digest of a protein sample are mass filtered and collisionally dissociated to yield product or fragment ions which are subsequently mass analyzed. The resulting product ion spectra can be used to determine the sequence of the parent peptide and, through a database search, the identity of the originating protein. This method of analysis can be automated and it is generally performed in conjunction with a real time liquid chromatography separation system. Although this methodology is quite powerful, it has been shown to have limitations when characterizing com-plex protein mixtures. Many of these limitations are associated with the serial na-ture of the MS/MS data acquisition process which limits both the qualitative and the quantitative reproducibility of the information generated. A new method of protein characterization of proteolytically-generated peptides has been developed which is based on coupling the alternate scanning of pep-tides at low and elevated energy1,2 with a robust accurate mass LC/MS data ac-quisition mode3. This mode of data acquisition is very high in duty cycle and pro-vides exact mass MS analysis of both the precursor and product ions for every de-tectable peptide. Chromatographic retention time is used to associate fragments to their corresponding precursors as a unique attribute of the Waters Protein Ex-pression System technology. Proprietary Protein Expression System Informatics software has been developed which allows this information to be processed in a quantitative and qualitative manner. This poster will focus on the qualitative identi-fication capabilities of this software. Poster TPR 357 (Geromanos et al., Towards Global Proteomics by Analysis of Exact Mass Retention Time Pairs: A proof-of-principle of the Quantitative Ion Mapping Technology) addresses the quantitative capabilities of the software and 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 Experiments) highlights the reproducibility of the Protein Expression System platform.

    Towards Global Proteomics, All Ions all the Time: A Proof-of-Principle of the Qualitative Ion Mapping Technology

    Jeffrey C. Silva1, Keith Richardson2, Phillip Young2, Richard Denny2, Kieran Neeson2, Therese McKenna2, Craig Dorschel1, Guo-Zhong Li1, Marc Gorenstein1, Timothy Riley1, and Scott Geromanos1 1Waters Corporation, Milford, MA USA, 2Waters Corporation, Manchester, UK

    Introduction

    Sample Preparation

    Data Processing

    Non-depleted human serum (Sigma, Cat. No. R9759) was tryptically digested according to a procedure employing a proprietary non-ionic detergent and in which cysteine residues are reduced with DTT and alkylated with iodoacetimide prior to digestion (see Poster TPY 458). The resulting samples contain 0.94 1.13 ug human serum protein per microliter.

    Data Collection

    A basic premise of the Protein Expression System Informatics software is that the exact mass MS data acquired at low energy is primarily composed of peptide precursor ion information and the exact mass MS data acquired at elevated en-ergy is composed primarily of corresponding peptide product or fragment ion in-formation. It is also understood that fragment ions associated with a particular parent ion are produced simultaneously with the parent ions as they elute from the reverse phase column. Protein Expression System Informatics filters the data acquired in the alternating scan experiment in a three-step process. The first step carefully aligns chroma-tographic retention time to associate fragment ions to each appropriate precursor ion (Figure 1). The second step compares the exact mass of each measured peptide precursor ion to a database containing the accurate mass of every pep-tide (including one missed cleavage) associated with every known protein in the proteome under study. Typically, peptides which are within an acceptable mass measurement tolerance (within +/- 5 ppm) are identified in a subset peptide data base (Figure 2). The accurate mass of each y and b fragment ion is calculated for each of the peptides in this subset database and the measured exact mass fragment ion information obtained in the first processing step is compared to this theoretical accurate mass information (Figure 3). A probability-based search model that takes into account the number, type, and mass accuracy of the meas-ured fragment ions is used to determine the best peptide sequence match. Figure 1. Figure 2.

    Figure 3.

    Figures 4 and 5 illustrate typical results from an alternating low and elevated collision energy LC/MS analysis of non-depleted human serum tryptic digest. The upper panel of Figure 4 illustrates the low energy base peak intensity chroma-togram and the lower panel shows a low energy exact mass spectrum extracted at the highlighted retention time of 72.390 minutes. Figure 5 illustrates the same information for the elevated energy function with the example spectrum extracted one scan later at 72.425 minutes. While many peptide ions co-elute during this moment in time, only a subset of the peptide ions apex at the same moment in time. Figure 6 shows the low energy exact mass chromatograms for singly and doubly charge state ions for 4 example peptides that co-elute in the chroma-tographic region around 72 minutes. The singly and doubly charged ions associ-ated with the same peptide clearly apex at the same chromatographic retention time. Figure 7 examines the high energy data in the same retention time region. The center panel shows the exact mass chromatograms of a number of high energy ions some of which are clearly unrelated to each other. The left panel shows the low energy exact mass chromatogram for m/z 872.54 precursor at 71.96 min-utes (lower trace) and six example high energy fragment ions that appear to be related since they chromatographically apex at the time. Similarly, the right hand panel shows six example high energy fragment ions that appear to be related to the low energy m/z 739.96 peptide at 72.60 minutes.

    Results and Discussion

    Figure 5.

    Figure 6. Figure 7.

    As noted in the data processing section, Protein Expression System Informatics uses this principle of chromatographic 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 (see Poster WPJ 161, Goren-stein et al. Statistical Study of Ions and Peptides Found In LC/MS/MS Analysis of Human Serum Digest and Poster TPR 354, Li et al.). Figure 8 illustrates this cleaning principle more graphically. The top panel shows the combined ele-vated energy spectra of all ions that elute at 72.425 minutes. The middle panel shows the elevated energy spectra after being cleaned of ions that do not co-apex with the low energy doubly charged peptide, m/z 872.54, at 71.96 min-utes. Similarly, the bottom panel shows the elevated energy spectra after removal of ions that do not co-apex with the low energy doubly charged peptide, m/z 739.96, at 72.60 minutes. In Table 1, the qualitative identification data filtering steps illustrated in Figures 2 and 3 have been applied to the 739.96 doubly charged peptide ion eluting at 72.60 minutes along with its chromatographically associated fragment ions. Each of the proteins listed in Table 1 contains a tryptic peptide sequence that has an accurate mass within plus or minus 5ppm of the 739.96 measured mass. In Tables 1 and 2, columns 5 and 6 indicate those corresponding b- and y-ions for each peptide, that are found in the elevated energy data to within plus or minus 10 ppm mass accuracy and plus or minus 0.05 minutes retention time. A 0 at a given amino acid position indicates the corresponding fragment ion was not found within the specified search tolerances, and a 1 at a given amino acid position indicates the corresponding fragment ion was found within the specified search tolerances. The measured exact mass fragment ion data associated with the 739.96 peptide parent ion has been compared to the theoretical y and b fragment ions associated with each of the candidate parent ions in Table 1. The measured fragment ions associated with the MYLGYEYVTAIR clearly provide con-vincing evidence, with twelve matching fragment ions, to indicate that the 739.96 doubly charged peptide originates from the Serotransferrin precursor pro-tein. Table 2, illustrates a similar analysis of the 872.54 doubly charged parent ion and its chromatographically associated fragment ions. In this example, there are 21 fragment ions that validate YAASSYLSLTPEQWK as the tryptic peptide to Ig lambda chain C. Table 3 illustrates the top 82 proteins identified in a tryptic digest of undepleted human serum using the Protein Expression System profiling strategy. Protein se-quence coverage is provided for each of the identified proteins. Figure 8.

    A novel alternating low and elevated energy exact mass LC/MS data acquisition strategy is proposed as an alternative to conventional data dependent LC/MS/MS analysis for protein identification. This strategy provides an enhanced duty cycle relative to data dependent MS/MS analysis and essentially yields MS and MS/MS like fragmentation information on all detectable peptide in a chromatographic analysis of a protein digest. Preliminary studies suggest that this new Waters Protein Expression System profiling strategy identifies more proteins with better sequence coverage than data dependent LC/MS/MS analysis. 1 Bateman, Robert Harold, et al. 2001. Methods and apparatus for mass spectrometery. United States patent application 20010052569 A1 December 20, 2001. 2 S Purvine, JT Eppel, EC Yi and DR Goodlett (2003) Proteomics 3: 847-850. 3 S Geromanos, A Dongre, G Opiteck, JC Silva (2003) Micromass Limited, PCT filing WO/054549.

    Table 1.

    Table 2.

    Table 3.

    Summary

    LC/MS System: Waters Protein Expression System comprised of the Waters CapLC Sys-tem 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 m 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 L/min, followed by 10 min. rinse (99% B) and 20 min. re-equilibration at initial conditions Injections of 10.0 L of the human serum sample were made directly onto the column. Mass spectral data were collected in alternating 1.8 second scans, with a 0.2 second inter-scan delay, at collision cell energies of 10 eV for the low energy mode and an energy ramp from 28 to 35 eV for the elevated scanning mode. The NanoLockSpray source was sampled every 10 seconds to obtain a scan of the exact mass standard ([Glu1]-Fibrinopeptide). The raw data were processed using functions in ProteinLynx Global SERVER and Protein Expression System Informatics software (Poster TPR 354, Li et al., A Novel Algorithm to Track Ions or Peptides Found by LC/MS in Multiple Injections of Multiple Complex Biologi-cal Samples). Additional data analysis was performed using Microsoft Excel and Spotfire DecisionSite.

    Collect all fragment ions

    Collect all precursor ions

    Data Acquisition ChromatographicAlignment

    Collect all fragment ions

    Collect all precursor ions

    Data Acquisition ChromatographicAlignment

    Collect all fragment ions

    Collect all precursor ions

    Data Acquisition ChromatographicAlignment

    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 (tryptic peptides)

    MS

    y1ynbnb1y1ynbnb1

    LE

    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

    y1ynbnb1y1ynbnb1

    EE

    100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z0

    100

    %

    080603_497HL_QuanExpSerial_03 812 (72.390) Cm (812) 1: TOF MS ES+ 610739.9572

    656.4526

    630.7704295.1562 490.3446

    872.5406

    807.0036873.5505

    1084.1528874.0437

    927.5532 1085.14451479.92001275.2264 1744.1146

    10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00Time0

    100

    %

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ BPI

    5.70e360.8324.5721.36

    7.09

    6.23

    5.16

    7.74 20.79

    10.52

    13.01 19.79

    29.00

    50.9130.85 42.91

    34.49 46.7060.18

    65.47 75.75

    73.60

    70.75

    77.96

    78.81 108.06106.06

    95.0089.8781.67

    103.2197.00

    112.84 118.05

    100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z0

    100

    %

    080603_497HL_QuanExpSerial_03 812 (72.390) Cm (812) 1: TOF MS ES+ 610739.9572

    656.4526

    630.7704295.1562 490.3446

    872.5406

    807.0036873.5505

    1084.1528874.0437

    927.5532 1085.14451479.92001275.2264 1744.1146

    10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00Time0

    100

    %

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ BPI

    5.70e360.8324.5721.36

    7.09

    6.23

    5.16

    7.74 20.79

    10.52

    13.01 19.79

    29.00

    50.9130.85 42.91

    34.49 46.7060.18

    65.47 75.75

    73.60

    70.75

    77.96

    78.81 108.06106.06

    95.0089.8781.67

    103.2197.00

    112.84 118.05

    Cs 1, 1744.12

    Cs 2, 1744.12

    Cs 1, 1630.01

    Cs 2, 1630.01

    Cs 1, 1478.95

    Cs 2, 1478.95

    Cs 1, 1311.92

    Cs 2, 1311.92

    70.00 70.50 71.00 71.50 72.00 72.50 73.00 73.50 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

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    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1744.115 0.05Da

    15771.96

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 872.541 0.05Da

    6.87e371.96

    73.32

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1630.014 0.05Da

    94.472.68

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 815.51 0.05Da

    3.75e372.68

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1478.95 0.05Da

    14472.60

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 739.957 0.05Da

    4.57e372.60

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1311.915 0.05Da

    14170.82

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 656.453 0.05Da

    7.94e370.82

    71.82

    Cs 1, 1744.12

    Cs 2, 1744.12

    Cs 1, 1630.01

    Cs 2, 1630.01

    Cs 1, 1478.95

    Cs 2, 1478.95

    Cs 1, 1311.92

    Cs 2, 1311.92

    70.00 70.50 71.00 71.50 72.00 72.50 73.00 73.50 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1744.115 0.05Da

    15771.96

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 872.541 0.05Da

    6.87e371.96

    73.32

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1630.014 0.05Da

    94.472.68

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 815.51 0.05Da

    3.75e372.68

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1478.95 0.05Da

    14472.60

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 739.957 0.05Da

    4.57e372.60

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1311.915 0.05Da

    14170.82

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 656.453 0.05Da

    7.94e370.82

    71.82

    Cs 1, 1744.12

    Cs 2, 1744.12

    Cs 1, 1630.01

    Cs 2, 1630.01

    Cs 1, 1478.95

    Cs 2, 1478.95

    Cs 1, 1311.92

    Cs 2, 1311.92

    70.00 70.50 71.00 71.50 72.00 72.50 73.00 73.50 74.00Time0

    100

    %

    70.00 70.50 71.00 71.50 72.00 72.50 73.00 73.50 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

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    0

    100

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    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1744.115 0.05Da

    157

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1744.115 0.05Da

    15771.96

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 872.541 0.05Da

    6.87e371.96

    73.32

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1630.014 0.05Da

    94.472.68

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 815.51 0.05Da

    3.75e372.68

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1478.95 0.05Da

    144

    71.96

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 872.541 0.05Da

    6.87e371.96

    73.32

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1630.014 0.05Da

    94.472.68

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 815.51 0.05Da

    3.75e372.68

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1478.95 0.05Da

    14472.60

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 739.957 0.05Da

    4.57e372.60

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 1311.915 0.05Da

    14170.82

    080603_497HL_QuanExpSerial_03 1: TOF MS ES+ 656.453 0.05Da

    7.94e370.82

    71.82

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21264.801 0.05Da

    112

    71.0770.07 74.28

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21101.748 0.05Da

    319

    71.07

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2988.631 0.05Da

    525

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2901.626 0.05Da

    278

    74.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2788.489 0.05Da

    695

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2687.429 0.05Da

    1.32e3

    73.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F1872.541 0.05Da

    6.87e3

    73.32

    72.00

    72.00

    72.00

    72.00

    72.00

    72.00

    71.96

    Precursor

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21264.801 0.05Da

    112

    71.0770.07 74.28

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21101.748 0.05Da

    319

    71.07

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2988.631 0.05Da

    525

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2901.626 0.05Da

    278

    74.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2788.489 0.05Da

    695

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2687.429 0.05Da

    1.32e3

    73.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F1872.541 0.05Da

    6.87e3

    73.32

    72.00

    72.00

    72.00

    72.00

    72.00

    72.00

    71.96

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21264.801 0.05Da

    112

    71.0770.07 74.28

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21101.748 0.05Da

    319

    71.07

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2988.631 0.05Da

    525

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2901.626 0.05Da

    278

    74.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2788.489 0.05Da

    695

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2687.429 0.05Da

    1.32e3

    73.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F1872.541 0.05Da

    6.87e3

    73.32

    72.00

    72.00

    72.00

    72.00

    72.00

    72.00

    71.96

    Precursor

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

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    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21014.657 0.05Da

    144

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2901.626 0.05Da

    278

    74.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2851.562 0.05Da

    35070.29 74.14

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2788.489 0.05Da

    695

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2722.506 0.05Da

    374

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2687.429 0.05Da

    1.32e3

    73.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2559.432 0.05Da

    189

    70.21 74.50

    72.57

    72.00

    72.57

    72.00

    72.57

    72.00

    72.57

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

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    0

    100

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    0

    100

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    0

    100

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    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21014.657 0.05Da

    144

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2901.626 0.05Da

    278

    74.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2851.562 0.05Da

    35070.29 74.14

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2788.489 0.05Da

    695

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2722.506 0.05Da

    374

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2687.429 0.05Da

    1.32e3

    73.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2559.432 0.05Da

    189

    70.21 74.50

    72.57

    72.00

    72.57

    72.00

    72.57

    72.00

    72.57

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    70.00 72.00 74.00Time0

    100

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    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21014.657 0.05Da

    144

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2901.626 0.05Da

    278

    74.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2851.562 0.05Da

    35070.29 74.14

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2788.489 0.05Da

    695

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2722.506 0.05Da

    374

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2687.429 0.05Da

    1.32e3

    73.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2559.432 0.05Da

    189

    70.21 74.50

    72.57

    72.00

    72.57

    72.00

    72.57

    72.00

    72.57

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21014.657 0.05Da

    144

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2901.626 0.05Da

    278

    74.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2851.562 0.05Da

    35070.29 74.14

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2788.489 0.05Da

    695

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2722.506 0.05Da

    374

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2687.429 0.05Da

    1.32e3

    73.21

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2559.432 0.05Da

    189

    70.21 74.50

    72.57

    72.00

    72.57

    72.00

    72.57

    72.00

    72.57

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21184.781 0.05Da

    322

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21071.702 0.05Da

    531

    70.07

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21014.657 0.05Da

    144

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2851.562 0.05Da

    35070.29 74.14

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2722.506 0.05Da

    374

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2559.432 0.05Da

    189

    70.21 74.50

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F1739.957 0.05Da

    4.57e372.60

    72.56

    72.56

    72.56

    72.56

    72.56

    72.56

    Precursor

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21184.781 0.05Da

    322

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21071.702 0.05Da

    531

    70.07

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21014.657 0.05Da

    144

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2851.562 0.05Da

    35070.29 74.14

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2722.506 0.05Da

    374

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2559.432 0.05Da

    189

    70.21 74.50

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F1739.957 0.05Da

    4.57e372.60

    72.56

    72.56

    72.56

    72.56

    72.56

    72.56

    Precursor

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21184.781 0.05Da

    322

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21071.702 0.05Da

    531

    70.07

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21014.657 0.05Da

    144

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2851.562 0.05Da

    35070.29 74.14

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2722.506 0.05Da

    374

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2559.432 0.05Da

    189

    70.21 74.50

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F1739.957 0.05Da

    4.57e372.60

    72.56

    72.56

    72.56

    72.56

    72.56

    72.56

    70.00 72.00 74.00Time0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    0

    100

    %

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21184.781 0.05Da

    322

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21071.702 0.05Da

    531

    70.07

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F21014.657 0.05Da

    144

    70.00

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2851.562 0.05Da

    35070.29 74.14

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2722.506 0.05Da

    374

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F2559.432 0.05Da

    189

    70.21 74.50

    080603_497HL_QuanExpSerial_03 Sm (SG, 2x3) F1739.957 0.05Da

    4.57e372.60

    72.56

    72.56

    72.56

    72.56

    72.56

    72.56

    Precursor

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Fragment

    Pro

    tein Ex

    pressio

    n Sy

    stem In

    form

    atics P

    rocessin

    g

    P

    r

    o

    t

    e

    i

    n

    E

    x

    p

    r

    e

    s

    s

    i

    o

    n

    S

    y

    s

    t

    e

    m

    I

    n

    f

    o

    r

    m

    a

    t

    i

    c

    s

    P

    r

    o

    c

    e

    s

    s

    i

    n

    g

    UncleanFragment Ion Data

    Clean 872.54++Fragment Ion Data

    Clean 739.96++Fragment Ion Data

    UncleanFragment Ion Data

    Clean 872.54++Fragment Ion Data

    Clean 739.96++Fragment Ion Data

    0100011000000000000010000000003.31743.8674WATHGVPSAVNSHPQRO94808 Glucosamine-fructose-6P. aminotransferase 2

    00100010000000000000001000003.01743.8669THLGLIMDGWNSMIRO94935 KIAA0851 protein

    1100000000000000000000000000002.51743.8660NYGILADATEQVGQHKQ9Y4J7 Fibroblast growth factor 2-interacting factor

    000000000000000000000010000000-4.01743.8548GGEPNFLPVTEEADIRO43736 Integral membrane protein 2A (E25 protein)

    000000000000000000000000003.41743.8676GFLLQNEFVGFMWRQ9GZU1 CDNA FLJ22449 fis clone HRC09609

    1111111111101001111111110000-1.61743.8588YAASSYLSLTPEQWKP01842 Ig lambda chain C regions

    00000000000000000000010000002.51743.8660TASHPQDYTVENLIRO75018 Leucocyte Immunoglobulin-like Receptor 6A/B

    100000000000000001000000000000-4.01743.8548SHPLQLTDDGGFSEIKQ9H0K9 Hypothetical 78 6 kDa protein

    0000000000000000000000000000-2.01743.8582NEEIVMPEEQTGLVRQ9H473 Golgi-spec. brefeldin A-resistance guanine nef-1

    1010010000000000000000000000-4.01743.8548LLEQSEWQPTNVDGKQ96GG7 Interferon regulatory factor 1

    00000000100000000010000000000000-2.01743.8582GDEEGVPAVVIDMSGLRP13796 L-plastin (Lymphocyte cytosolic protein 1)

    1000000000000000100000000000004.81743.8701DSSEIPGALWHIYAGKQ9P2G7 KIAA1380 protein

    100000000000100011000000000000-2.01743.8582MATEGLHENETLASLKO14775 Guanine nucleotide-binding protein beta subunit 5

    y"-ions(y1.yn-1)

    b-ions(b1...bn-1)

    Delta (ppm)Actual MH+SequenceDescription

    0100011000000000000010000000003.31743.8674WATHGVPSAVNSHPQRO94808 Glucosamine-fructose-6P. aminotransferase 2

    00100010000000000000001000003.01743.8669THLGLIMDGWNSMIRO94935 KIAA0851 protein

    1100000000000000000000000000002.51743.8660NYGILADATEQVGQHKQ9Y4J7 Fibroblast growth factor 2-interacting factor

    000000000000000000000010000000-4.01743.8548GGEPNFLPVTEEADIRO43736 Integral membrane protein 2A (E25 protein)

    000000000000000000000000003.41743.8676GFLLQNEFVGFMWRQ9GZU1 CDNA FLJ22449 fis clone HRC09609

    1111111111101001111111110000-1.61743.8588YAASSYLSLTPEQWKP01842 Ig lambda chain C regions

    00000000000000000000010000002.51743.8660TASHPQDYTVENLIRO75018 Leucocyte Immunoglobulin-like Receptor 6A/B

    100000000000000001000000000000-4.01743.8548SHPLQLTDDGGFSEIKQ9H0K9 Hypothetical 78 6 kDa protein

    0000000000000000000000000000-2.01743.8582NEEIVMPEEQTGLVRQ9H473 Golgi-spec. brefeldin A-resistance guanine nef-1

    1010010000000000000000000000-4.01743.8548LLEQSEWQPTNVDGKQ96GG7 Interferon regulatory factor 1

    00000000100000000010000000000000-2.01743.8582GDEEGVPAVVIDMSGLRP13796 L-plastin (Lymphocyte cytosolic protein 1)

    1000000000000000100000000000004.81743.8701DSSEIPGALWHIYAGKQ9P2G7 KIAA1380 protein

    100000000000100011000000000000-2.01743.8582MATEGLHENETLASLKO14775 Guanine nucleotide-binding protein beta subunit 5

    y"-ions(y1.yn-1)

    b-ions(b1...bn-1)

    Delta (ppm)Actual MH+SequenceDescription

    00000000000011000000003.01478.7427YVVTSVSWWSHKP55884 Eukaryotic translation initiation factor 3 subunit 9 (eIF-3 eta)

    1000000000000000000000-5.01478.7308VDIIENQVMDFRP21266 Glutathione S transferase Mu 3

    10000000000000100000000.31478.7387NSWPSQISLQYRQ9UNI1 Elastase 1 precursor

    0000000000000000000000003.01478.7427DLWHAAFFLSGSKQ9H099 Hypothetical 78 5 kDa protein

    1000000000000000000000-5.01478.7308AMDLVQEEFLQRQ9HB07 MYG1 protein

    00000000000000000000000.01478.7382WLSMNVLMLDEKP50440 Glycine amidinotransferase, mitochondrial precursor

    000000000000000000000000-0.61478.7373SALPYENIDSEIKO60522 Colon cancer antigen NY-CO-45 (Fragment)

    000100000000000000000000-0.61478.7373NSETFPTILEEAKO60610 Diaphanous protein homolog 1

    1011111011101110000000-2.31478.7348MYLGYEYVTAIRP02787 Serotransferrin precursor (Siderophilin)

    00000000000000000000000000-5.01478.7308MTSGNISVSWPATKQ9BXU7 Ubiquitin carboxyl-terminal hydrolase 26

    00000000000000000000000000-0.61478.7373ESDDGVVVYVAPTKQ9NVE3 CDNA FLJ10787 fis clone NT2RP4000481

    1000000000000000000000000.31478.7387AGFNSYAELLTHRQ9H8J4 CDNA FLJ13564 fis clone PLACE1008201

    100000000000100000000000002.61478.7420MEVSGDPGIPALHRQ96KJ4 Similar to pre-pro-megakarycyte potentiating factor

    y"-ions(y1.yn-1)

    b-ions(b1..bn-1)

    Delta (ppm)Actual MH+SequenceDescription

    00000000000011000000003.01478.7427YVVTSVSWWSHKP55884 Eukaryotic translation initiation factor 3 subunit 9 (eIF-3 eta)

    1000000000000000000000-5.01478.7308VDIIENQVMDFRP21266 Glutathione S transferase Mu 3

    10000000000000100000000.31478.7387NSWPSQISLQYRQ9UNI1 Elastase 1 precursor

    0000000000000000000000003.01478.7427DLWHAAFFLSGSKQ9H099 Hypothetical 78 5 kDa protein

    1000000000000000000000-5.01478.7308AMDLVQEEFLQRQ9HB07 MYG1 protein

    00000000000000000000000.01478.7382WLSMNVLMLDEKP50440 Glycine amidinotransferase, mitochondrial precursor

    000000000000000000000000-0.61478.7373SALPYENIDSEIKO60522 Colon cancer antigen NY-CO-45 (Fragment)

    000100000000000000000000-0.61478.7373NSETFPTILEEAKO60610 Diaphanous protein homolog 1

    1011111011101110000000-2.31478.7348MYLGYEYVTAIRP02787 Serotransferrin precursor (Siderophilin)

    00000000000000000000000000-5.01478.7308MTSGNISVSWPATKQ9BXU7 Ubiquitin carboxyl-terminal hydrolase 26

    00000000000000000000000000-0.61478.7373ESDDGVVVYVAPTKQ9NVE3 CDNA FLJ10787 fis clone NT2RP4000481

    1000000000000000000000000.31478.7387AGFNSYAELLTHRQ9H8J4 CDNA FLJ13564 fis clone PLACE1008201

    100000000000100000000000002.61478.7420MEVSGDPGIPALHRQ96KJ4 Similar to pre-pro-megakarycyte potentiating factor

    y"-ions(y1.yn-1)

    b-ions(b1..bn-1)

    Delta (ppm)Actual MH+SequenceDescription

    % Coverage Description Median Mass Error61 Zinc alpha 2 glycoprotein precursor 1.112 Vitamin D-binding protein precursor 3.426 Very low density lipoprotein receptor 3.714 Vascular endothelial growth factor 2.658 u plasminogen activator precursor 6.045 t plasminogen activator precursor 4.338 Sex steroid binding globulin precursor validated 5.248 Serum paraoxonase arylesterase 1 5.772 Serum albumin precursor 3.771 Serotransferrin precursor 2.538 Retinoid acid induced protein 1 4.618 Platelet factor 4 precursor 6.636 Plasminogen activator inhibitor 1 9.722 Plasminogen 3.741 Phospholipase C precursor 0.016 Phospholipase A2 precursor 6.617 Leukocyte elastase 4.459 Lactotransferrin precursor 5.544 Kininogen LMW precursor 7.319 Interleukin 2 precursor 5.245 Interleukin 18 binding protein precursor 8.426 Interleukin 12B precursor 5.918 Inter-alpha-trypsin inhibitor heavy chain H1 2.442 Insulin like growth factor II precursor 1.045 Insulin like growth factor IA/B precursor 7.651 IgE Fc receptor II low affinity 8.156 Ig MU 3.168 Ig lambda 1.748 Ig kappa 0.753 Ig heavy chain V III 1.658 Ig heavy chain V I 1.463 Ig heavy chain V 2.044 Ig gamma 3.430 Ig alpha 2.346 Hypothetical 137 2 kDa protein Fragment 4.752 Hemopexin 1.463 Hemoglobin zeta chain 6.155 Hemoglobin gamma G chain 0.155 Hemoglobin gamma A chain 0.258 Hemoglobin delta chain 3.065 Hemoglobin beta chain 1.4

    % Coverage Description Median Mass Error58 Hemoglobin alpha chain 0.234 Heat shock 70 kDa protein 1 6.352 Haptoglobulin-2 2.855 Haptoglobulin-1 3.039 Haptoglobin related protein 4.448 Factor VII 4.438 Complement factor MASP 3 6.250 Complement factor H related protein 1 5.052 Complement factor H precursor H factor 1 5.749 Complement component C9 precursor 7.658 Complement component C8 beta chain precursor 2.938 Complement component C7 precursor 4.929 Complement component C6 precursor 4.434 Complement component C5 precursor 5.847 Complement component C4 precursor 4.468 Complement component C3 precursor 2.741 Collagen alpha 3 IX chain precursor 5.236 Coagulation factor XIIa 4.541 Coagulation factor XIa 3.755 Coagulation factor Xa 4.740 Coagulation factor VIIa 6.363 Chain A Human Serum Transferrin N Lobe 2.045 Ceruloplasmin 3.530 Cathepsin K precursor 6.038 Cathepsin D precursor 1.115 C reactive protein precursor 4.833 Apolipoprotein H precursor 3.264 Apolipoprotein A-II 2.863 Apolipoprotein A-I 2.862 Antithrombin III 3.139 Antileukoproteinase 1 precursor 6.230 Angiotensinogen 6.153 Alpha-2-macroglobulin 1.042 Alpha-2-HS-glycoprotein 5.138 Alpha-2-antiplasmin 3.245 Alpha-1-microglobulin inter alpha trypsin inhibitor 3.236 Alpha-1B-glycoprotein 2.866 Alpha-1-antitrypsin precursor 1.265 Alpha-1-acid glycoprotein 2 precursor 3.454 Alpha-1-acid glycoprotein 1 precursor 2.439 Alpha 3 type IV collagen 6.2

    10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00Time0

    100

    %

    080603_497HL_QuanExpSerial_03 2: TOF MS ES+ BPI

    3.53e373.42

    38.527.13

    6.20

    5.13

    21.8920.82

    7.77

    9.9119.04

    22.11 28.11

    24.54 33.60

    60.9442.88

    46.73

    51.80 60.29

    58.51 67.86

    78.49

    78.99106.10

    89.5581.71 95.18 103.24 108.09 117.66112.30 119.79

    100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z0

    100

    %

    080603_497HL_QuanExpSerial_03 812 (72.425) Cm (812) 2: TOF MS ES+ 235267.1570

    136.0944 1071.7020295.1492

    687.4291

    366.1824 670.4338809.5603

    988.6307

    1184.77981185.7744

    1478.93411386.79211186.7974 1613.9867 1745.0836

    10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00Time0

    100

    %

    080603_497HL_QuanExpSerial_03 2: TOF MS ES+ BPI

    3.53e373.42

    38.527.13

    6.20

    5.13

    21.8920.82

    7.77

    9.9119.04

    22.11 28.11

    24.54 33.60

    60.9442.88

    46.73

    51.80 60.29

    58.51 67.86

    78.49

    78.99106.10

    89.5581.71 95.18 103.24 108.09 117.66112.30 119.79

    100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z0

    100

    %

    080603_497HL_QuanExpSerial_03 812 (72.425) Cm (812) 2: TOF MS ES+ 235267.1570

    136.0944 1071.7020295.1492

    687.4291

    366.1824 670.4338809.5603

    988.6307

    1184.77981185.7744

    1478.93411386.79211186.7974 1613.9867 1745.0836

    ASMS 2004