Introduction to mass spectrometry-based protein identification and quantification Austin Yang, Ph.D. Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003 Mar 13;422(6928):198-207. Review. Mueller LN, Brusniak MY, Mani DR, Aebersold R An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data. J Proteome Res. 2008 Jan;7(1):51-61.
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Introduction to mass spectrometry-based protein identification and quantification
Introduction to mass spectrometry-based protein identification and quantification. Austin Yang, Ph.D. Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003 Mar 13;422(6928):198-207. Review. Mueller LN, Brusniak MY, Mani DR, Aebersold R - PowerPoint PPT Presentation
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Introduction to mass spectrometry-based protein identification and quantification
Austin Yang, Ph.D.
Aebersold R, Mann M.Mass spectrometry-based proteomics.Nature. 2003 Mar 13;422(6928):198-207. Review.
Mueller LN, Brusniak MY, Mani DR, Aebersold RAn assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data.J Proteome Res. 2008 Jan;7(1):51-61.
The typical proteomics experiment consists of five stages
Mass spectrometers used in proteome research.
Monoistopic Mass = 1155.6
Average Mass = 1156.3 (calculated)
As shown in Figure 1. the monoisotoptic mass of this compound is 1155.6. For a given compound the monoisotopic mass is the mass of the isotopic peak whose elemental composition is composed of the most abundant isotopes of those elements. The monoisotopic mass can be calculated using the atomic masses of the isotopes.
The average mass is the weighted average of the isotopic masses weighted by the isotopic abundances. The average mass can be calculated using the atomic weights of the elements.
500 peptides with masses closest to that of the parent ion are retrieved from a protein database. Computer generates a theoretical MS/MS Spectrum for each peptide sequence (SEQ1, 2, 3, 4, …)
(Experimental MS/MS Spectrum)
Theoretical MS/MSspectra
Step 1.Determine Parent
Ion molecular mass
Step 2.
Step 3.Experimental Spectrum is compared with each theoretical spectra and correlation scores are assigned.
Step 4.Scores are ranked andProtein Identifications are made based on these cross correlation scores.
ZSA-charge assignment
Unified Scoring Function
Prot APeptide 1
Peptide 2
Prot BPeptide 3
Peptide 4
Peptide 5
Prot
Prot
Prot
Prot
in the sample(enriched for ‘multi-hit’ proteins)
not in the sample(enriched for ‘single hits’)Prot
Peptide 6
Peptide 7
Peptide 8
Peptide 9
Peptide10
+
++
+
+
5correct (+)
Amplification of False Positive Error Rate from Peptide to Protein Level
Peptide Level: 50% False Positives
Protein Level: 71% False Positives
Quantitative Mass Spec Analysis
1. Relative Quantitationa. ICAT: Isotope-Coded Affinity Tagsb. Digestion with Oxygen-18 Waterc. Spectra Counting and Non-labeling Methodology 2. Absolute Quantitation
Cysteine C3H5NOS 103.00918
Carboxymethyl Cys C5H7NO3S 161.01466 58.00548
Alkylation of Cysteine Residue
Mascot Example Slides
ICAT
Trypsin Digestion with Oxygen18 and Oxygen16 Water
• Database: bovine• Fixed modifications : Carbamidomethyl (C)• Peptide Tolerance: 8 Da (required for O18
labeling)• Fragment Tolerance: 0.2 Da• Quantification Method: 18O corrected multiplex
MS/MS Database Search Example
• Data: BSA onespectra.mgf (one spectra)• Database: bovine• Fixed modifications: Carboxymethyl(C + 58.01)• Varied modifications: Oxidatation(M)• Peptide Mass Tolerance : 0.1 Da • Fragment Mass Tolerance: 0.1 Da • http://www.matrixscience.com/help/
fragmentation_help.html
MS2 mixture example
• Data: mixture10spectra.mgf• Database: yeast• Fixed modifications : Carbamidomethyl (C+57.02) • Variable modifications : Oxidation (M)• Peptide Mass Tolerance : 0.1 Da • Fragment Mass Tolerance: 0.1 Da
Home Work1. You will have to download your datasets from the following url:http://134.192.153.220/GPLS716 a. Identification of phosphorylation site : Data:BIG3021307.RAW.mgf Recommend parameters: Database: human. Variable Modification: Phospho(ST) Fixed modification: Carboamidomethyl(C).
b. Quantificaiton of oxygen-18/oxygen-16 digested BSA
Data: 18O_BSA_500fmol_1to5_071013.RAW.mgf.
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1. Your name and ID in the subject of your email 2. Search parameters