924.5113 965.4456 100 1593.7693 1586.8064 1630.7738 1657.7953 1689.7865 0 799.0 833.0566 842.4926 870.5201 878.4913 A mass spectrum 6.3E+4 80 70
2700.0 Basically measures mass Adapted from google Components Adapted from an Analytical chemistry textbook Ionization process MALDI ESI Matrix Assisted Laser Desorption Ionization ElectroSpray Ionization Nobel prize in Chemistry, 2002 MALDI Matrix Assisted Laser Desorption Ionization
ESI Electro Spray Ionization Mass analyzers several designs Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207 GPCL inventory ABI Voyager DE PRO, walk-up use 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 Time-of-flight (TOF) analyzers MALDI TOF Voyager DE PRO
ESI TOF Ultimate 3000 with micrOTOF MALDI TOF - principle KE = zeV = 1mv2 2 MS of serum albumin ESI TOF MALDI TOF Tandem mass spectrometer MALDI TOF/TOF MS and MS/MS Ion Trap MS, MS2, MS3, .MSn Quadrupole-q-TOF
ESI QqTOF installation phase. FT MS bottom line ..Resolution and mass accuracy FWHM Full width at half maxima of a peak Resolution and mass accuracy m measured at 50% peak height is the Full Width at Half Maxima (FWHM) R= M m R
M m = resolution = mass of the peak of interest = width in daltons of the peak Mass accuracy is measured as parts per million value ppm = 106m = 106 M R outline Mass spectrometers Protein identification Quantitative proteomics Protein-protein interactions Peptide Mass Fingerprinting - PMF Database entry NCBI
From: http://gobi.ym.edu.tw/course/mass/2004-0325.pdf Informatics Search engines Mascot, Matrix Science Sequest, Thermoelectron Free-ware Protein prospector (http://prospector.ucsf.edu/) TPP tools (http://tools.proteomecenter.org/TPP.php) Database searching using
MASCOT Overview of the experiment Submission of data to MASCOT webserver 1D SDS PAGE of proteins Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207 10 0 699.0 20 1081.5479 1159.2 1249.6954 1399.7751 1554.7437
1619.4 Mass (m/z) Mass to charge ratio (m/z) 2079.6 2555.2903 2493.3501 2458.3052 2262.0557 1895.0386 1763.7820 1730.7723 1687.8691 1640.0277
841.5205 789.5378 %Intensity Intensity Mass spectrum 4700 Reflector Spec #1 MC=>TR[BP = 1479.9, 15779] 1.6E+4 2539.8 3000.0 Peak list Compiled from the mass spectra
Mass list Mass list and intensity Submitted to the search engine http:// www.matrixscience.com/ Mascot scoring A frequency factor matrix, F, is created, in which each row represents an interval of 100 Da in peptide mass, and each column an interval of 10 kDa in intact protein mass. As each sequence entry is processed, the appropriate matrix elements fi,j are incremented so as to accumulate statistics on the size distribution of peptide masses as a function of protein mass. The elements of F are then normalised by dividing the elements of each 10 kDa column by the largest value in that column to give the Mowse factor matrix M: After searching the experimental mass values against a calculated peptide mass database, the score for each entry is calculated according to: Where MProt is the molecular weight of the entry and the product term is calculated from the Mowse factor elements for each match between the experimental data and peptide masses calculated from the entry.
List of common contaminants Trypsin autolysis peptides Matrix peaks Keratin from skin, hair Other contaminants Protein Identification Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207 Tandem mass spectrum http://qbab.aber.ac.uk Tandem mass spectrum 4700 MS/MS Precursor 1570.7 Spec #1 MC[BP = 175.1, 3106] 175.1326 100 3105.9
1445.2834 1559.9417 1570.2634 1551.7002 1658.0 Tandem mass spectra (MS/MS) can be used for peptide sequencing Database Searching Peptide Mass Fingerprinting Sequence tag approach De novo sequencing inspect raw data http://qbab.aber.ac.uk Mascot Search Results Search title : SampleSetID: 362, AnalysisID: 567, MaldiWellID: 15790, SpectrumID: 17225, Path=\Mani\102004\New Analysis 1
Database : NCBInr 20040606 (1846720 sequences; 611532004 residues) Timestamp : 20 Oct 2004 at 14:52:50 GMT Top Score : 681 for gi|180570, creatine kinase [Homo sapiens] Probability Based Mowse Score Score is -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 75 are significant (p<0.05). Top hits from Mascot Search there are multiple accession numbers for the same protein Accession 1. gi|180570 2. gi|21536286 3. gi|33304149 4. gi|125292 5. gi|180572 6. gi|125295 7. gi|180555 8. gi|203476
LGFSEVELVQMVVDGVK LGFSEVELVQMVVDGVK GTGGVDTAAVGGVFDVSNADR GTGGVDTAAVGGVFDVSNADR RGTGGVDTAAVGGVFDVSNADR RGTGGVDTAAVGGVFDVSNADR FPAEDEFPDLSAHNNHMAK LAVEALSSLDGDLAGRYYALK LRFPAEDEFPDLSAHNNHMAK LRFPAEDEFPDLSAHNNHMAK TDLNPDNLQGGDDLDPNYVLSSR TDLNPDNLQGGDDLDPNYVLSSR HGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSR HGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSR Creatine kinase B is the highest scoring protein Match to: gi|21536286 ; Score: 681 Nominal mass (Mr): 42591; Calculated pI Creatine kinase
From 2D gels .to MALDI or ESI MS Control Test Pool Cy3 Cy5 Image analysis with Delta2D, Decodon Quantitate Export spot list to robotic picker ..its high-throughput 1st Dimension - Isoelectric focussing 2nd Dimension SDS PAGE Spot picking Trypsin gel digest
Colorectal cancer markers Isolate Nuclear Matrix Mass spectral analysis MS In-gel Tryptic digest m/z MS/MS Database Search Tumor specific markers CC3, CC4, CC5, CC6a, CC6b m/z 2D 1
D Immunoblotting Immunohistochemistry Protein Identified Validation Yes No de novo sequencing Balasubramani et al., Cancer Res., 2006 Shotgun proteomics Adapted from Aebersold, R.; Mann, M. Nature 2003, 422, 198-207 typical workflow to identify biomarkers that distinguish indolent versus aggressive forms of cancer..
Group A, Indolent Group B, Aggressive Fractionate Fractionate Eg. Immunodeplete, subcellular Eg. Immunodeplete, subcellular Tryptic peptides Tryptic peptides Label with iTRAQ reagent 115 Label with iTRAQ reagent 116 Combine labeled digests LC fractionate MS and MS/MS Protein ID and Quantitate
Sample handling In-solution Isoelectric focussing HPLC 1D or 2D LC MALDI Protein-protein interaction studies Immunoaffinity pull-downs Tandem affinity purification GPCL Billy W Day Paul Wood Mirunalni Thangavelu Tamanna Sultana Emanuel M Schreiber Chris Bolcato Chris Myers Patrick Miller
Robert Wolfe definitions The amu is defined as 1/12th the mass of one neutral 6C12 atom Amu is also called the dalton 1 amu =1/12 ( 12g 12C/mol 12C 6.0221 x 1023 atoms 12C 1.6605 x 10-24 g/atom 12C C/mol 12 Isotopic species of M (M + H)+
Chiang Rai Rajabhat University. Assignment 11. Vocabulary2. Demonstration Problem 3. ... Purchases Journal, and Cash Receipts Journal with Cash Dr., Sales Discounts Dr., Sales Cr.. and Accounts Receivable Cr. ... A check mark in parentheses is placed immediately below the...
Rough Order Of Magnitude (ROM) Estimate of Effort Classification These are rough order of magnitudes and do not include project ramp-up and testing effort (SIT & UAT) and only assumes unit testing. Actual estimates could vary significantly depending on requirements.
LYU0002 Wireless Cyber Campus Supervisor: Prof. Michael R. Lyu Student: Jang Kim Fung Tang Ho Man Presentation Outline Introduction Project Aim Progress of 1st Semester Problem faced & Solution Progress of 2nd Semester Future Work Conclusion Introduction (1) Traditional Campus...
And finally there is the glad, simple, unhindered offering of your gift to God. Teaching about Adultery. 27 "You have heard the commandment that says, 'You must not commit adultery.' 28 But I say, anyone who even looks at a...
Clinical Research Librarian, Health Science Center Libraries. Rolando Garcia-Milian. Basic Biomedical Sciences Librarian, Health Science Center Libraries. Hannah F. Norton. Liaison and Reference Librarian, Health Science Center Libraries. Cecilia E. Botero. Associate Dean of the George A. Smathers Libraries and....
Are Alma / Primo users _____ Sandra: Agenda, Rationalizing, Envisioning part 1 (slides 3-13) Highlights of the vision and how we got to the collaboration outcomes (what does this actually look like in a library system?) Title slide: Relate to...