1. 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.

2. The typical proteomics experiment consists of five stages

3. Mass spectrometers used in proteome research.

4. 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. www.ionsource.com

5. Electrospray Ionization (ESI) Multiple charging Multiple charging – More charges for larger molecules MW range > 150 kDa MW range > 150 kDa Liquid introduction of analyte Liquid introduction of analyte – Interface with liquid separation methods, e.g. liquid chromatography – Tandem mass spectrometry (MS/MS) for protein sequencing 5007009001100 mass/charge (m/z) 20+ 19+ 18+ 17+ 16+ 15+ 14+ 21+ 22+ highly charge droplets MS ESI

6. Origin of the ES Spectra of Peptides H H H H 4+4+ H H H 3+3+ H 2+2+ H 1+1+ H m/z = (M r +4H)/4 m/z = (M r +3H)/3 m/z = (M r +2H)/2 m/z = (M r +H) 1+1+ 2+2+ 3+3+ 4+4+ Rel. Inten. m/z ES-MS

7. b1b1 b2b2 b3b3 y1y1 y2y2 y3y3 LFG K Relative Intensity m/z FLGK ++ FLGK ++ FLGK ++ CID FLGK ++ FLGK ++ FLGK ++ b1b1 b2b2 b3b3 y3y3 y2y2 y1y1 FLGK ++ FLGK + Theoretical CID of a Tryptic Peptide KGLF MS/MS Spectrum Parent ions (464.29) Daughter ions Non-dissociated Parent ions

8. Peptide Sequencing by LC/MS/MS

9. Web addresses of some representative internet resources for protein identification from mass spectrometry data

10. Data Mining through SEQUEST and PAULA DatabaseSearch Time Yeast ORFs (6,351 entries) 52 sec: 0.104 sec/s Non-redundant protein (100k entries) 3500 min: EST (100K entries, 3-frames) 5-10,000 min:

11. STEP 1. SEQUEST Algorithm (Experimental MS/MS Spectrum) 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/MS spectra 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 and Protein Identifications are made based on these cross correlation scores. ZSA-charge assignment Unified Scoring Function

12. Prot A Peptide 1 Peptide 2 Prot B Peptide 3 Peptide 4 Peptide 5 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 + + + + + 5 correct (+) Amplification of False Positive Error Rate from Peptide to Protein Level Peptide Level: 50% False Positives Protein Level: 71% False Positives

13. Quantitative Mass Spec Analysis 1. Relative Quantitation a. ICAT: Isotope-Coded Affinity Tags b. Digestion with Oxygen-18 Water c. Spectra Counting and Non-labeling Methodology 2. Absolute Quantitation

15. Cysteine C3H5NOS 103.00918 Carboxymethyl Cys C5H7NO3S 161.01466 58.00548 Alkylation of Cysteine Residue

16. Mascot Example Slides ICAT

17. Trypsin Digestion with Oxygen18 and Oxygen16 Water

18. Johri et al. Nature Reviews Microbiology 4, 932 – 942 (December 2006) | doi:10.1038/ nrmicro1552 Absolute Quantification

19. Public Web Server http://www.matrixscience.com/search_form_select.html Class Data Download: http://134.192.153.220/GPLS716 Local Web Server http://134.192.153.220/mascot Username: GPLS716 Password: GPLS716 http://134.192.153.220/mascot

20. MS1 PMF(peptide mass fingerprinting) Search Example Data: testms1.txt, 210 MS1 peaks Database: bovine Fixed modifications : Carboxymethyl (C) Variable modifications : Oxidation (M) Peptide Tolerance: 0.1 Da Monoisotopic mass Mass Value: Mr

21. Quantification Search Example Data: 18O_BSA_100fmol_1to5_01_071018.RAW.mgf Database: bovine Fixed modifications : Carbamidomethyl (C) Peptide Tolerance: 8 Da (required for O18 labeling) Fragment Tolerance: 0.2 Da Quantification Method: 18O corrected multiplex

22. 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/fragmen tation_help.html

23. 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

24. Home Work 1. You will have to download your datasets from the following url:http://134.192.153.220/GPLS716http://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. Submit your search results in pdf or html format to the following email address: proteomicsumb@gmail.com; Please include the following information when you submit your homework proteomicsumb@gmail.com 1. Your name and ID in the subject of your email 2. Search parameters 3. A short summary of your search results. Questions: Contact Yunhu Wan, email: ywan@som.umaryland.edu Phone number: 8-2031