![Page 1: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/1.jpg)
MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein
Digests
Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein
Mass Spectrometry Data Center
![Page 2: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/2.jpg)
Library searching in not new
Organize for Reuse
![Page 3: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/3.jpg)
MS Library Searching
• Hertz, Hites and Biemann Anal. Chem. (1971).
• PBM: McLafferty, Hertel, Villwock Org. Mass Spectrom. (1974).
• SISCOM: Damen, Henneberg, Weimann, Anal. Chem. Acta (1978).
• INCOS: Sokolow, Karnofsky, Gustafson , Finnigan Application Report 2 (March 1978).
• Stein, Scott J. Amer. Soc. Mass Spectrom., (1994).
![Page 4: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/4.jpg)
‘Dot Product’(cosine of ‘angle’ between a pair of spectra)
• Measured = f(m/z abundance) • Reference = f(m/z abundance)• f(abundance) : Weight as you like
RM
MRSum over all peaks in common
Normalize
![Page 5: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/5.jpg)
Traditional GC/MS Library Search
![Page 6: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/6.jpg)
Variability Depends on S/N
~7,000 Radiodurans
Peptides, LCQ
(PNNL/NCRR)Medians
![Page 7: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/7.jpg)
Library Searching for Peptides
• LIBQUEST (Yates)– Yates et al, Anal. Chem., 1998, 70, 3557
• X!Hunter (Beavis)– Craig et al, J. Proteome Res., 2006, 5, 1843
• BiblioSpec (MacCoss)– Frewen et al., Anal. Chem. 2006, 78, 5678
• Spectral Comparison (Kearney) – Liu et al, Proteome Science 2007, 5:3
• SpectraST (Aebersold)– Lam et al., Proteomics 2007 6, 655-667
• NIST Peptide Ion Fragmentation Library– June 2006 release (US-HUPO – March 2004)
![Page 8: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/8.jpg)
![Page 9: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/9.jpg)
Why Spectrum Libraries?
• More sensitive
• Better scoring
• Faster
• Annotation
• Unrestricted precursor ion
![Page 10: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/10.jpg)
Fraction of MS/MS Spectra Identified vs S/N
0.001
0.01
0.1
1
1 10 100 1000 10000
S/N
Fra
cti
on
ID
ed
All Peptides
HSA Peptides
HSA-OMSSA
Identification by Spectrum Matching is More Sensitive than by Spectrum/Sequence Matching
Simple Protein Mix
![Page 11: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/11.jpg)
Spectrum/Spectrum Scores are More Robust than Sequence/Spectrum Scores
Sequence score
99% Confidence
![Page 12: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/12.jpg)
0.005/s vs. 6.2/s per query spectrum
Matching Spectra is Faster than Matching Sequence
![Page 13: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/13.jpg)
Reference Library Building
• Extract identified spectra from sequence search
– Multiple search engines
– Instrument-class specific
• Create ‘consensus’ spectra
– Two or more matching spectra, also save best
• Assign probability of being correct
– Refine confidence starting from decoy FDR
– Classify peptides – tryptic, missed cleavage, semi, mods
• Create searchable spectral library
– Resolve conflicts, add annotation
![Page 14: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/14.jpg)
Three Classes of Libraries
I. Conventional Target Identification
– Peptides (Proteins)
II. Identifiable
– By unconventional searching
III. Not Identifiable
– Account for all recurring spectra
– QA/QC
![Page 15: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/15.jpg)
I. OMSSA overlap with MS/MS Library Search
747 1350 353
34K6/06
318 1752 833
78K6/07
Identified spectra (1% FDR) for 1-D Yeast NCI/CPTAC – Vanderbilt
![Page 16: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/16.jpg)
Semitryptic
Tryptic bad miss
Tryptic missed cleavage Tryptic
Identified Spectra: Yeast - 1 D
![Page 17: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/17.jpg)
II. Identify What we CanDerive Class-specific FDR
• Tryptic– Simple– Expected missed cleavages– Unexpected missed cleavages
• Semitryptic (cleaved tryptic)– No missed cleavage
• In source (with parent at same retention)• In sample
– Missed cleavage• In source (with parent)• In sample (obey rules)• Uncommon – reject
• Others …
![Page 18: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/18.jpg)
Atypical Peptide Ionsuse Sequence Search Method
• Tryptic only with many mods• Less common: Methylation, Phosphorylation, …• Artifacts: Na, K, Carbamyl• InsPecT/Pevzner (Unidentified, +70)
• High charge states, >2 missed cleavages
• Use class specific score thresholds
![Page 19: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/19.jpg)
HSA/Fibrinogen/Transferrin Mix
6124 Consensus Peptide Spectra, IT, Qtof, TofTof
Ion Trap Peptide Ions: 1300 HSA, 1100 Fibrinogen, 700 Transferrin
![Page 20: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/20.jpg)
contiguous = tryptic, exploded = semitryptic
Bad missMissed
'Insample'
Insource
Unknown modBad miss
Missed
Simple
Identified Peptide Spectra - Simple Protein Mix
![Page 21: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/21.jpg)
III. Library ofRecurring, Unidentified Spectra
• Create consensus spectra– From similar spectra from an experiment
• Combine from multiple experiments
• Identify spectra in other experiments– QA/QC: Artifacts, in standards, …– Apply other sequencing methods
![Page 22: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/22.jpg)
Assign all Spectra• Identified Spectrum
– Matches library peptide or unidentified spectrum– Subset of peaks match library spectrum (impure)– Similar to a matched spectrum (cluster)
• Not a Peptide– Low S/N
• Maximum/Median <15– High charge state (many large peaks)
• Proteins, large fragments, …– One dominant peak
• Stable ion, not peptide– Singly charged (high/low abund < 1.2)
• Probable artifact, lower probability of identification– Narrow m/z range
• Peptide?
![Page 23: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/23.jpg)
exploded = identified, contiguous = unidentified
Peptide?
1+ No ID
OtherLow S/N
NoID Lib/Impure
NoID Lib
Peptide/Impure
Peptide
Spectrum Classification - Yeast - 1D
![Page 24: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/24.jpg)
exploded = identified, contiguous = unidentified
Spectrum Classification - Simple Protein Mix
Peptide?
1+ NoID
NarrowComplex
Dominant PeakLow S/N
NoID Lib/ClusterNoID Lib/Impure
NoID lib
Pep/Cluster
Pep/Impure
Peptide
![Page 25: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/25.jpg)
Library Pipeline of the Future
assigned
No ID No IDPep.Lib
Unass.Lib
unassigned
No ID
Garbage filter
Sequence Search,
De Novo,Theoretical
Spec,Similarity, ...No ID
assigned
Mass spectrometer
![Page 26: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/26.jpg)
NCI/NIH - CPTAC:Clinical Proteomic Technology Assessment
for Cancer
http://proteomics.cancer.gov
Technology assessment; develop standard protocols and clinical reference sets; and evaluate methods to ensure data reproducibility.
Broad Institute of MIT and Harvard, Memorial Sloan-Kettering Cancer Center, Purdue University,
University of California, San Francisco,, and Vanderbilt University School of Medicine.
NCI grants (U24CA126476-01, U24CA126485-01, U24CA126480-01, U24CA126477-01, and U24CA126479-01).
![Page 27: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/27.jpg)
![Page 28: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/28.jpg)
RT: 10.01 - 70.06
15 20 25 30 35 40 45 50 55 60 65 70
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
Re
la
tive
A
bu
nd
an
ce
NL: 6.73E6
TIC F: ITMS + c ESI Full ms [300.00-2000.00] MS NCI_study2_021607_sample1B228_vial_03
Run-to-Run Chromatographic Reproducibility
RT: 9.99 - 70.13
10 15 20 25 30 35 40 45 50 55 60 65 70
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
Re
la
tive
A
bu
nd
an
ce
NL: 5.53E6
TIC F: ITMS + c ESI Full ms [300.00-2000.00] MS NCI_study2_021607_sample1B33_vial_01
![Page 29: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/29.jpg)
CPTAC_STUDY2_WEEK1_1B144_01 2/27/2007 11:31:04 AMHPLC: CPTAC - Dilute 150x - Inj 2 ul
RT: 0.00 - 100.00
0 10 20 30 40 50 60 70 80 90 100
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
75.15493.21
46.50516.2741.98
543.2548.07569.75
56.53409.54
63.95575.3141.54
749.799.06
401.1166.44500.81
3.95363.79
12.11401.11
33.69322.18
25.07337.68
80.95528.38
85.52426.73
88.72445.12
NL: 6.63E8
TIC F: FTMS + p NSI Full ms [300.00-2000.00] MS CPTAC_STUDY2_WEEK1_1B144_01
CPTAC_STUDY2_WEEK1_1B144_01 #1745 RT: 16.39 P: + NL: 6.89E6F: FTMS + p NSI Full ms [300.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
345.52
517.78371.10
540.29692.30 741.20 869.43 1679.171612.381497.98 1925.52
nw_022207o_liebler_study2_Vanderbilt_... 2/24/2007 6:08:26 AM
RT: 0.00 - 100.00
0 10 20 30 40 50 60 70 80 90 100
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
12.65390.14
11.79401.11
13.35421.06
45.28647.29
31.61387.4529.16
722.32 40.65409.543.43
313.0249.31
547.3223.46
358.8540.14
660.0653.26
507.3015.17
588.3368.38
671.8256.75749.38 70.47
682.7095.22
313.0290.34
313.0283.06
313.02
NL: 4.30E7
TIC F: FTMS + p NSI Full ms [300.00-2000.00] MS nw_022207o_liebler_study2_Vanderbilt_Orib2_week1_1B035_070224060826
No scan(s) match the scan filter.
20070511_CPTAC_1B100 5/11/2007 10:26:15 AM
RT: 0.00 - 100.00
0 10 20 30 40 50 60 70 80 90 100
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bun
dan
ce
67.05493.21
42.27722.32 45.38
395.2458.53
575.31 78.46671.82
41.92492.75 55.91
500.7533.29749.792.79
319.1071.35
829.3879.61
454.6930.64
371.1084.49
319.1190.86
673.3614.78
371.1024.32
371.109.45
371.10
NL:1.08E8
TIC F: FTMS + p NSI Full ms [300.00-2000.00] MS 20070511_CPTAC_1B100
No scan(s) match the scan filter.
IN_LTQm_041907_1B274_02 4/20/2007 11:21:41 PM
RT: 0.00 - 100.00
0 10 20 30 40 50 60 70 80 90 100
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
dan
ce
39.78647.55
68.00682.96
63.29672.12
10.71493.27
1.17444.98
46.11547.69
35.05660.35 76.11
992.8662.31
556.9632.59
409.8577.97
673.64 85.691133.34
52.60749.65
3.98538.07
31.90501.74
16.74722.60
21.81516.71
87.16840.73 95.80
835.36
NL: 5.50E6
Base Peak m/z= 400.00-2000.00 F: ITMS + c ESI Full ms MS IN_LTQm_041907_1B274_02
IN_LTQm_041907_1B274_02 #3160 RT: 34.26 P: + NL: 1.05E5F: ITMS + c ESI Full ms
400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
Inte
nsity
318.42
864.14
535.49472.81635.43
451.36751.54352.85 874.75 1069.11 1280.621171.71 1451.95 1553.29 1897.561725.84
liebler_Vanderbilt_1B121_100 2/24/2007 11:17:00 PM
RT: 0.00 - 100.00
0 10 20 30 40 50 60 70 80 90 100
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Ab
un
dan
ce
47.00647.53
69.41672.1651.41
547.79
42.50409.84
71.26682.89
58.37749.72
65.71484.14
31.29722.72
33.23493.0329.63
441.01 83.82673.6975.32
544.3823.82
538.085.02
401.1619.28
749.9599.02
406.4594.31
461.97
NL: 6.09E6
Base Peak m/z= 400.00-2000.00 F: ITMS + c NSI Full ms [300.00-2000.00] MS liebler_Vanderbilt_1B121_100
No scan(s) match the scan filter.
BroadOrbitrap
VandyOrbitrap
NYUOrbitrap
INCAPSLTQ
NCI_study2_021607_sample1B228_vial_03 2/16/2007 8:45:21 PM sample 1B
RT: 0.00 - 100.00
0 10 20 30 40 50 60 70 80 90 100
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Ab
un
dan
ce
32.21492.49
40.30647.47
27.35722.5022.63
537.94 63.77671.96
66.20682.8835.90
660.212.69
536.2947.13
547.6919.46749.98 49.18
500.93 56.48829.583.66
444.4273.99
992.737.98508.17
78.50674.14
86.001133.70
96.96435.96
NL: 2.51E6
Base Peak m/z= 400.00-2000.00 F: ITMS + c ESI Full ms MS NCI_study2_021607_sample1B228_vial_03
NCI_study2_021607_sample1B228_vial_03 #3205 RT: 34.26 P: + NL: 5.70E4F: ITMS + c ESI Full ms
400 600 800 1000 1200 1400 1600 1800 2000
m/z
0
10000
20000
30000
40000
50000
Inte
nsi
ty
566.86
849.57
390.88
631.19
354.17 860.65530.80680.41 952.15731.19 1625.481237.471022.05 1364.34 1521.92 1696.42 1865.63 1951.58
NISTLTQ
VandyLTQ
0703141B289 3/15/2007 12:58:07 PM
RT: 0.00 - 100.00
0 10 20 30 40 50 60 70 80 90 100
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
39.43575.73
33.33410.00
41.64547.76
24.18516.87
42.88501.15
23.07722.47 47.81
481.7025.84718.0618.27
408.1053.04498.08
58.16426.98 95.61
432.1770.94615.41
65.81419.06
84.48419.13
74.13419.12
1.01445.03
15.131451.05
NL: 5.80E5
Base Peak m/z= 400.00-2000.00 F: ITMS + c NSI Full ms [300.00-2000.00] MS 0703141B289
No scan(s) match the scan filter.
PurdueLTQ
YICENQDSISSK
Lab-to-Lab Chromatography
![Page 30: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/30.jpg)
HSA_CAM_SigmaA9511_5H_8MS2_m2_10de_040406_05
![Page 31: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/31.jpg)
Measures of Reproducibility
• Identified ions– Unique peptides, Ions, Spectrum counts
• Unidentified components– Classify by type, link to origin
• Ion cluster analysis– MS1 linked to MS2
• Chromatography– Time evolution of ion clusters
![Page 32: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/32.jpg)
Ion Component Analysis
![Page 33: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/33.jpg)
Ion Component Analysis (Yeast)
1E-3 0.01 0.1
10
100
1000
Oversampling
Relative Component Intensity
Co
un
ts
Components All MS2 Sampled Peptides
Undersampling
![Page 34: MS/MS Libraries of Identified Peptides and Recurring Spectra in Protein Digests Lisa Kilpatrick, Jeri Roth, Paul Rudnick, Xiaoyu Yang, Steve Stein Mass](https://reader035.vdocuments.net/reader035/viewer/2022081420/56649ebf5503460f94bca97b/html5/thumbnails/34.jpg)
1E-4 1E-3 0.01 0.1
10
100
1000
Nu
mb
er
of C
om
po
ne
nts
Component Intensity
Components in Replicate Runs
total
sampled
identified
▲▼ run 1,2 ■ in both