Proteomics and mass spectrometry

Proteomics and mass spectrometry l.jpg
1 / 51
949 days ago, 247 views
PowerPoint PPT Presentation
Layout. Mass spectrometers Protein ID Quantitative proteomics Protein-protein associations. . A mass range. . Fundamentally measures mass. Adjusted from google . Segments

Presentation Transcript

Slide 1

Proteomics and mass spectrometry Manimalha Balasubramani

Slide 2

Outline Mass spectrometers Protein ID Quantitative proteomics Protein-protein communications

Slide 3

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

Slide 4

Basically measures mass Adapted from google

Slide 5

Components… Adapted from an Analytical science course book

Slide 6

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

Slide 7

MALDI – Matrix Assisted Laser Desorption Ionization

Slide 8

ESI – Electro Spray Ionization

Slide 9

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

Slide 10

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

Slide 11

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

Slide 12

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

Slide 13

MS of serum egg whites ESI TOF MALDI TOF

Slide 14

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

Slide 15

Ion Trap MS, MS 2 , MS 3 , … .MS n

Slide 16

Quadrupole-q-TOF ESI QqTOF

Slide 17

… establishment stage… . FT MS

Slide 18

… main concern… ..Resolution and mass exactness…

Slide 19

FWHM Full width at half maxima of a pinnacle

Slide 20

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

Slide 21

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

Slide 22

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

Slide 23

Peptide Mass Fingerprinting - PMF Database section NCBI From:

Slide 24

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

Slide 25

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

Slide 26

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

Slide 27

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

Slide 28

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

Slide 29

Slide 30

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

Slide 31

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

Slide 32

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

Slide 33

Tandem mass range

Slide 34

Tandem mass range

Slide 35

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

Slide 36

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).

Slide 37

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

Slide 38

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

Slide 39

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%

Slide 40

diagram Mass spectrometers Protein ID Quantitative proteomics Protein-protein cooperations

Slide 41

Quantitative Proteomics Sample arrangement

Slide 42

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

Slide 43

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

Slide 44

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

Slide 45

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

Slide 46

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..

Slide 47

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

Slide 48

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

Slide 49

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

Slide 50

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

Slide 51

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