Mueller LN, Brusniak MY, Mani DR, Aebersold R 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.

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. www.ionsource.com

Atomic Masses and Abundances for a Subset of Naturally Occurring Biologically Relevant Isotopes % A+1 A+2 A+3 A+4 12C 12 98.93(8) 13C 13.0033548378(10) 1.07(8) 14C 14.003241988(4) - 1H 1.0078250321(4) 99.9885(70) 2H 2.0141017780(4) 0.0115(70) 3H 3.0160492675(11) 14N 14.0030740052(9) 99.632(7) 15N 15.0001088984(9) 0.368(7) 16O 15.9949146221(15) 99.757(16) 17O 16.99913150(22) 0.038(1) 18O 17.9991604(9) 0.205(14) 32S 31.97207069(12) 94.93(31) 33S 32.97145850(12) 0.76(2) 34S 33.96786683(11) 4.29(28) 36S 35.96708088(25) 0.02(1) . 19F 18.99840320(7) 100 23Na 22.98976967(23) 39K 38.9637069(3) 93.2581(44) 40K 39.96399867(29) 0.0117(1) 41K 40.96182597(28) 6.7302(44) 31P 30.97376151(20) 35Cl 34.96885271(4) 75.781(4) 37Cl 36.96590260(5) 24.22(4) 55Mn 54.9380496(14) 54Fe 53.9396148(14) 5.845(35) 56Fe 55.9349421(15) 91.754(36) 57Fe 56.9353987(15) 2.119(10) 58Fe 57.9332805(15) 0.282(4) 63Cu 62.9296011(15) 69.17(3) 65Cu 64.9277937(19) 30.83(3) 79Br 78.9183376(20) 50.69(7) 81Br 80.916291(3) 49.31(7) 127I 126.904468(4)

Peak Abundance, “Mass Crossover” and Calibration

The Nobel Prize in Chemistry 2002 "for the development of methods for identification and structure analyses of biological macromolecules" "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules" John Fenn Koichi Tanaka

Mass Spectrometry: A method to “weigh” molecules Other information can be inferred from a weight measurement. Post-translational modifications Molecular interactions Shape Sequence Physical dimensions etc... A simple measurement of mass is used to confirm the identity of a molecule, but it can be used for much more……

Matrix-assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) Analyzer detector high voltage v1 m1 v2 m2 v3 m3 MALDI sample laser drift region m1 m2 m3

Electrospray: Generation of aerosols and droplets “Wings to Molecular Elephants”

Electrospray Ionization (ESI) Multiple charging More charges for larger molecules MW range > 150 kDa Liquid introduction of analyte Interface with liquid separation methods, e.g. liquid chromatography Tandem mass spectrometry (MS/MS) for protein sequencing ESI MS high voltage highly charge droplets 20+ 19+ 18+ 21+ 17+ 16+ 22+ 15+ 14+ 500 700 900 1100 mass/charge (m/z)

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

Theoretical CID of a Tryptic Peptide + + + + F L G K + + F L G K b3 y1 F L G K + + Parent ions + + F L G K F L G K CID b2 y2 + F L G K + + + + F L G K F L G K b1 y3 Non-dissociated Parent ions Daughter ions y1 y2 y3 b1 b2 b3 K G L F MS/MS Spectrum Relative Intensity L F G K m/z (464.29)

Peptide Sequencing by LC/MS/MS

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

Data Mining through SEQUEST and PAULA Database Search 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:

Determine Parent Ion molecular mass SEQUEST Algorithm Theoretical MS/MS spectra Step 1. Determine Parent Ion molecular mass Step 2. STEP 1. SEQ 1 SEQ 2 SEQ 3 SEQ 4 500 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) ZSA-charge assignment Step 4. Scores are ranked and Protein Identifications are made based on these cross correlation scores. Step 3. Experimental Spectrum is compared with each theoretical spectra and correlation scores are assigned. STEP 3. Unified Scoring Function (Experimental MS/MS Spectrum)

Amplification of False Positive Error Rate from Peptide to Protein Level + Prot A Peptide 1 in the sample (enriched for ‘multi-hit’ proteins) Peptide 2 Prot B + Peptide 3 + 5 correct (+) Peptide 4 Peptide 5 Prot Peptide 6 not in the sample (enriched for ‘single hits’) + Peptide 7 Prot Prot Peptide 8 Prot Peptide 9 Prot + Peptide10 Peptide Level: 50% False Positives Protein Level: 71% False Positives

Quantitative Mass Spec Analysis 1. Relative Quantitation a. SILAC and iTRAQ b. Digestion with Oxygen-18 Water c. Spectra Counting and Non-labeling Methodology 2. Absolute Quantitation

Trypsin Digestion with Oxygen18 and Oxygen16 Water

Limitation of SILAC

Multiplexed Isobaric Tagging Technology (iTRAQ) Philip L. Ross, et al. Molecular & Cellular Proteomics 3:1154–1169, 2004.

Release of 114 and 117 Reporter Ions Parent Ion Regular CID to obtain sequence Low mass cut-off and no reporter ion High Energy Collision Cell to quantify and sequence

Protein name 117/114 ratio Num of pep PSD93 2.829 5 PSD95 2.021 21 Loading 10ug 9 salt cuts online 2D_LC_MS/MS 962 proteins are quantified Protein name 117/114 ratio Num of pep PSD93 2.829 5 PSD95 2.021 21 PSD95-AP1 1.764 2 GABA alpha 1.365 GABA beta 2.087 3 NR2B 1.813 4 AMPA1 2.092 7 AMPA2 1.921 11 AMPA4 1.902 NR1 1.658 6 Expected ratio

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

Public Web Server http://www. matrixscience. com/search_form_select Public Web Server http://www.matrixscience.com/search_form_select.html Class Data Download: http://10.90.157.112/GPLS716 Local Web Server http://10.90.157.112/mascot Username: GPILS Password: GPILS

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

Quantification Search Example Note: Save link as; Save this file to the desktop) 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 Peptide Charge: Mr Quantification Method: 18O corrected multiplex 32

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

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

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 Work 1. You will have to download your datasets from the following url:http://10.90.157.112/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_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 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