Proteomics and mass spectrometry

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Layout. Mass spectrometers Protein ID Quantitative proteomics Protein-protein associations. . A mass range. . Fundamentally measures mass. Adjusted from google . Segments

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Proteomics and mass spectrometry Manimalha Balasubramani

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Outline Mass spectrometers Protein ID Quantitative proteomics Protein-protein communications

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1258.5603 100 1303.7007 6.3E+4 90 80 70 60 878.4913 % Intensity 1031.5374 983.4860 50 1254.5614 1114.5428 1657.7953 40 842.4926 1586.8064 30 1232.5907 1964.8882 924.5113 1153.5334 20 1035.5696 965.4456 833.0566 1315.5780 1280.5370 1074.5405 2120.9883 1360.7209 1191.6130 1689.7865 2518.1062 1800.9324 1395.7062 1848.9419 1475.7374 870.5201 1630.7738 2211.0520 1593.7693 2169.9207 2393.0823 2439.0872 2021.9116 10 0 799.0 1179.2 1559.4 1939.6 2319.8 2700.0 Mass (m/z) A mass range

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Basically measures mass Adapted from google

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Components… Adapted from an Analytical science course book

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Ionization prepare M atrix A ssisted L aser D esorption I onization MALDI E lectroSpray I onization ESI Nobel prize in Chemistry, 2002

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MALDI – Matrix Assisted Laser Desorption Ionization

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ESI – Electro Spray Ionization

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Mass analyzers – a few plans Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

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GPCL stock ABI Voyager DE PRO, stroll up utilize ABI 4700 Proteomics Analyzer Thermoelectron LCQ Deca with Surveyor HPLC ABI Qstar Elite with Ultimate 3000 HPLC Bruker micrOTOF with Ultimate 3000 HPLC Bruker 12 Tesla FTMS with Ultimate 3000 HPLC

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Time-of-flight (TOF) analyzers MALDI TOF Voyager DE PRO ESI TOF Ultimate 3000 with micrOTOF

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MALDI TOF - standard KE = zeV = 1 m v 2

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MS of serum egg whites ESI TOF MALDI TOF

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Tandem mass spectrometer MALDI TOF/TOF MS and MS/MS

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Ion Trap MS, MS 2 , MS 3 , … .MS n

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Quadrupole-q-TOF ESI QqTOF

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… establishment stage… . FT MS

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… main concern… ..Resolution and mass exactness…

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FWHM Full width at half maxima of a pinnacle

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Resolution and mass precision Δ m measured at half pinnacle stature is the Full Width at Half Maxima (FWHM) R = M Δ m R = determination M = mass of the pinnacle of intrigue Δ m = width in daltons of the pinnacle

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Mass precision is measured as parts per million esteem ppm = 10 6 Δ m = 10 6 M R

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plot Mass spectrometers Protein recognizable proof Quantitative proteomics Protein-protein cooperations

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Peptide Mass Fingerprinting - PMF Database section NCBI From:

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Informatics Search motors Mascot, Matrix Science Sequest, Thermoelectron Free-product Protein miner ( TPP instruments (

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Database seeking utilizing MASCOT Overview of the examination Submission of information to MASCOT webserver

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1D SDS PAGE of proteins Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

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Mass range Intensity Mass to charge proportion (m/z)

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Peak list Compiled from the mass spectra Mass rundown Mass rundown and force Submitted to the web index

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A recurrence figure grid, F, is made, in which each line speaks to an interim of 100 Da in peptide mass, and every segment an interim of 10 kDa in place protein mass. As each grouping section is handled, the suitable framework components fi,j are increased to collect insights on the size appropriation of peptide masses as an element of protein mass. The components of F are then standardized by isolating the components of every 10 kDa segment by the biggest incentive in that section to give the Mowse consider framework M: After seeking the trial mass qualities against an ascertained peptide mass database, the score for every passage is figured by: Where MProt is the atomic weight of the section and the item term is computed from the Mowse consider components for each match between the test information and peptide masses ascertained from the passage. Mascot scoring

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List of regular contaminants Trypsin autolysis peptides Matrix tops Keratin from skin, hair Other contaminants

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Protein Identification Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

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Tandem mass range

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Tandem mass range

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Tandem mass spectra (MS/MS) can be utilized for peptide sequencing Database Searching Peptide Mass Fingerprinting Sequence label approach De novo sequencing inspect crude information

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Mascot Search Results Search title : SampleSetID: 362, AnalysisID: 567, MaldiWellID: 15790, SpectrumID: 17225, Path= \ Man i \ 102004 \ New Analysis 1 Database : NCBInr 20040606 (1846720 successions; 611532004 deposits) Timestamp : 20 Oct 2004 at 14:52:50 GMT Top Score : 681 for gi|180570 , creatine kinase [Homo sapiens] Probability Based Mowse Score is - 10*Log(P), where P is the likelihood that the watched match is an irregular occasion. Protein scores more prominent than 75 are huge (p<0.05).

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Top hits from Mascot Search – there are different increase numbers for a similar protein

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Search gives back a bunch of proteins with the same coordinating peptides

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Creatine kinase B is the most astounding scoring protein Match to: gi|21536286 ; Score: 681 Creatine kinase - B [Homo sapiens] Nominal mass (M r ): 42591 ; Calculated pI esteem: 5.34 Observed Mass & pI: 43kd, 6.2-6.27 Sequence Coverage: 46%

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diagram Mass spectrometers Protein ID Quantitative proteomics Protein-protein cooperations

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Quantitative Proteomics Sample arrangement

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From 2D gels … .to MALDI or ESI MS Control Test Pool Cy3 Cy5 Image investigation with Delta2D, Decodon Quantitate Export spot rundown to mechanical picker

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..its high-throughput… 1 st Dimension - Isoelectric focussing 2 nd Dimension – SDS PAGE Spot picking Trypsin gel process

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Mass otherworldly examination Isolate Nuclear Matrix MS In-gel Tryptic process m/z MS/MS Database Search Tumor particular markers CC3, CC4, CC5, CC6a, CC6b Protein Identified m/z 2D Validation Immunoblotting 1D Yes No Immunohistochemistry all over again sequencing Colorectal growth markers Balasubramani et al., Cancer Res., 2006

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Shotgun proteomics Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207

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Group An, Indolent Group B, Aggressive Fractionate Eg. Immunodeplete, subcellular Fractionate Eg. Immunodeplete, subcellular Tryptic peptides Tryptic peptides Label with iTRAQ reagent 116 Label with iTRAQ reagent 115 Combine marked condensations LC fractionate MS and MS/MS Protein ID and Quantitate … run of the mill work process to recognize biomarkers that recognize lethargic versus forceful types of disease..

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Sample dealing with In-arrangement Isoelectric focussing 1D or 2D LC MALDI HPLC

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Protein-protein collaboration considers Immunoaffinity pull-downs Tandem partiality sanitization

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GPCL Billy W Day Paul Wood Mirunalni Thangavelu Tamanna Sultana Emanuel M Schreiber Chris Bolcato Chris Myers Patrick Miller Robert Wolfe

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definitions The amu is characterized as 1/12 th the mass of one unbiased 6 C 12 molecule Amu is additionally called the dalton 1 amu =1/12 ( 12g 12 C/mol 12 C 6.0221 x 10 23 particles 12 C/mol 12 C 1.6605 x 10 - 24 g/iota 12 C

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Isotopic types of (M + H) + (M + 1H)/1H + (M + 2H) 2+ (M + 2H)/2H + (M + 3H) 3+ (M + 3H)/3H +