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Proteomics Informatics David Fenyő
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Course Information
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Protein Identification and Quantitation
Samples Peptides Mass Spectrometry Quantity intensity m/z Identity
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Central Dogma of Molecular Biology
Transcription Replication Translation Modification P
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X X Central Dogma of Molecular Biology Slow Fast P Transcription
Replication Slow Degradation Translation X Fast Degradation Modification P X
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Motivating Example: Protein Regulation
GRB7 ERBB4 Breast Cancer ERBB2 ERBB2 GRB7 ERBB4 ERBB2 ERBB2 GRB7 ERBB4 ERBB2 ERBB2
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Motivating Example: Protein Complexes
Alber et al., Nature 2007
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Motivating Example: Signaling
Choudhary & Mann, Nature Reviews Molecular Cell Biology 2010
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Identified and Quantified Proteins
Mass Spectrometry Based Proteomics Lysis Fractionation Digestion Mass spectrometry Peak Finding Charge determination De-isotoping Integrating Peaks Searching MS Identified and Quantified Proteins
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Ion Source Mass Analyzer Detector Mass Spectrometry intensity
mass/charge
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y b Mass Spectrometry Mass Analyzer 1 Frag-mentation Detector
Ion Source Mass Analyzer 2 y b
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Example data – ESI-LC-MS/MS
m/z m/z % Relative Abundance 100 250 500 750 1000 [M+2H]2+ 762 260 389 504 633 875 292 405 534 907 1020 663 778 1080 1022 MS/MS Time
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Information Content in a Single Mass Measurement
Human 10 8 6 Avg. #of matching peptides 4 3 2 1 #of matching peptides Tryptic peptide mass [Da] S. cerevisiae 10 8 6 Avg. #of matching peptides 4 3 2 1 #of matching peptides Tryptic peptide mass [Da]
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Compare, score, test significance Identified peptides and proteins
Protein Identification by Mass Spectrometry Samples Peptides MS/MS Protein DB Compare, score, test significance Identified peptides and proteins
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Repeat for all proteins Compare, Score, Test Significance
Tandem MS – Database Search Sequence DB Lysis Fractionation Pick Protein Digestion LC-MS Pick Peptide Repeat for all proteins MS/MS All Fragment Masses all peptides Repeat for MS/MS Compare, Score, Test Significance
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Search Results
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Search Results Most proteins show very reproducible peptide patterns
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Search Results
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Compare, Score, Test Significance
Spectrum Library Search Spectrum Library Lysis Fractionation Digestion LC-MS/MS Pick Spectrum all spectra Repeat for MS/MS Compare, Score, Test Significance Identified Proteins
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Interpretation of Mass Spectra
K L E D F G S m/z % Relative Abundance 100 250 500 750 1000
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Interpretation of Mass Spectra
K L E D F G S K 1166 L 1020 E 907 D 778 663 534 405 F 292 G 145 S 88 b ions m/z % Relative Abundance 100 250 500 750 1000
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Interpretation of Mass Spectra
K L E D F G S 147 K 1166 L 260 1020 E 389 907 D 504 778 633 663 762 534 875 405 F 1022 292 G 1080 145 S 88 y ions b ions m/z % Relative Abundance 100 250 500 750 1000
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Interpretation of Mass Spectra
K L E D F G S 147 K 1166 L 260 1020 E 389 907 D 504 778 633 663 762 534 875 405 F 1022 292 G 1080 145 S 88 y ions b ions m/z % Relative Abundance 100 250 500 750 1000 [M+2H]2+ 762 260 389 504 633 875 292 405 534 907 1020 663 778 1080 1022
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Interpretation of Mass Spectra
K L E D F G S 147 K 1166 L 260 1020 E 389 907 D 504 778 633 663 762 534 875 405 F 1022 292 G 1080 145 S 88 y ions b ions m/z % Relative Abundance 100 250 500 750 1000 [M+2H]2+ 762 260 389 504 633 875 292 405 534 907 1020 663 778 1080 1022
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Interpretation of Mass Spectra
K L E D F G S 147 K 1166 L 260 1020 E 389 907 D 504 778 633 663 762 534 875 405 F 1022 292 G 1080 145 S 88 y ions b ions m/z % Relative Abundance 100 250 500 750 1000 [M+2H]2+ 762 260 389 504 633 875 292 405 534 907 1020 663 778 1080 1022 113 113
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Interpretation of Mass Spectra
K L E D F G S 147 K 1166 L 260 1020 E 389 907 D 504 778 633 663 762 534 875 405 F 1022 292 G 1080 145 S 88 y ions b ions m/z % Relative Abundance 100 250 500 750 1000 [M+2H]2+ 762 260 389 504 633 875 292 405 534 907 1020 663 778 1080 1022 129 129
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Interpretation of Mass Spectra
K L E D F G S 147 K 1166 L 260 1020 E 389 907 D 504 778 633 663 762 534 875 405 F 1022 292 G 1080 145 S 88 y ions b ions m/z % Relative Abundance 100 250 500 750 1000 [M+2H]2+ 762 260 389 504 633 875 292 405 534 907 1020 663 778 1080 1022
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Interpretation of Mass Spectra
K L E D F G S 147 K 1166 L 260 1020 E 389 907 D 504 778 633 663 762 534 875 405 F 1022 292 G 1080 145 S 88 y ions b ions m/z % Relative Abundance 100 250 500 750 1000 [M+2H]2+ 762 260 389 504 633 875 292 405 534 907 1020 663 778 1080 1022
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Interpretation of Mass Spectra
K L E D F G S 147 K 1166 L 260 1020 E 389 907 D 504 778 633 663 762 534 875 405 F 1022 292 G 1080 145 S 88 y ions b ions m/z % Relative Abundance 100 250 500 750 1000 [M+2H]2+ 762 260 389 504 633 875 292 405 534 907 1020 663 778 1080 1022
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De Novo Sequencing Sequences consistent with spectrum
Amino acid masses 762 100 875 [M+2H]2+ % Relative Abundance 633 292 405 260 389 534 1022 504 663 778 907 1020 1080 250 500 750 1000 m/z Mass Differences Sequences consistent with spectrum
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Significance Testing False protein identification is caused by random matching An objective criterion for testing the significance of protein identification results is necessary. The significance of protein identifications can be tested once the distribution of scores for false results is known.
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C I Protein Quantitation by Mass Spectrometry Sample i Protein j Lysis
ij Protein j Lysis Peptide k Fractionation Digestion MS I LC - MS ik
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Protein Quantitation by Mass Spectrometry
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Protein Quantitation by Mass Spectrometry
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Protein Quantitation by Mass Spectrometry
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Protein Quantitation by Mass Spectrometry
Light Heavy Lysis Assumption: All losses after mixing are identical for the heavy and light isotopes and Fractionation Digestion Sample i Protein j Peptide k LC-MS MS H L Oda et al. PNAS 96 (1999) 6591 Ong et al. MCP 1 (2002) 376
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Protein Quantitation MS MS MS/MS MS/MS LC-MS Digestion Fractionation
Shotgun proteomics LC-MS Targeted MS 1. Records M/Z 1. Select precursor ion MS MS Digestion 2. Selects peptides based on abundance and fragments Fractionation 2. Precursor fragmentation MS/MS MS/MS Lysis 3. Protein database search for peptide identification 3. Use Precursor-Fragment pairs for identification Data Dependent Acquisition (DDA) Uses predefined set of peptides
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Compare, score, test significance Identified peptides and proteins
Proteogenomics Samples Peptides MS/MS Protein DB Compare, score, test significance Identified peptides and proteins
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Proteogenomics Next-generation sequencing of the genome Samples
and transcriptome Samples Peptides MS/MS Sample-specific Protein DB Compare, score, test significance Identified peptides and proteins
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Proteogenomics Non-Tumor Sample Genome sequencing
Identify germline variants Genome sequencing RNA-Seq Tumor Sample Identify alternative splicing, somatic variants and novel expression TCGAGAGCTG TCGATAGCTG Exon 1 Exon 2 Exon 3 Variants Alt. Splicing Novel Expression Exon X Fusion Genes Gene X Gene Y Tumor Specific Protein DB Reference Human Database (Ensembl)
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Proteogenomics ERBB2 Breast Cancer Breast
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Proteogenomics ERBB2 Breast Cancer Breast Ovarian Cancer
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Posttranslational Modifications
Peptide with two possible modification sites Matching MS/MS spectrum Intensity m/z Which assignment does the data support? 1, or 2, or 1 and 2?
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Protein Interactions Digestion Mass spectrometry Identification E F A
B Digestion Mass spectrometry Identification
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Data Analysis - Normalization
Normalized: mean=0, std=1 Raw Data
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Data Analysis - Normalization
Normalized 3 replicates Normalized 3 replicates + one more replicate a few months later
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Data Analysis
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FDA calls them “in vitro diagnostic multivariate assays”
Molecular Markers A molecular signature is a computational or mathematical model that links high-dimensional molecular information to phenotype or other response variable of interest. FDA calls them “in vitro diagnostic multivariate assays”
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