泛 用 蛋 白 質 體 學 之 質 譜 儀 資 料 分 析 平 台 的 建 立 與 應 用 Universal Mass Spectrometry Data Analysis Platform for Quantitative and Qualitative Proteomics
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1 泛 用 蛋 白 質 體 學 之 質 譜 儀 資 料 分 析 平 台 的 建 立 與 應 用 Universal Mass Spectrometry Data Analysis Platform for Quantitative and Qualitative Proteomics 2014 Training Course Wei-Hung Chang ( 張 瑋 宏 ) ABRC, Academia Sinica Principle Investigator: Dr. Yet-Ran Chen ( 陳 逸 然 ) ABRC, Academia Sinica
2 Mass Spectrometry-Based Proteomics Nature, 2003, 422, p Nature Genetics, 2003, 33, p Topics: Global Profiling of Proteome Identification of Unknown Proteins Quantification of Proteins Study of Post-Translational Modifications (PTMs) Study of Protein-Protein Interaction
3 The Feature of Mass Spectrometer High Sensitivity can detect molecules in trace level High Throughput couple the on-line chromatography for complete sample analysis High Quantification Accuracy high linear dynamic range support stable isotopic quantification High Identification Confidence reliable and published ID algorithms
4 Proteomic Workflow Protein Biological Question Acquire Biological Sample Specific Physiological Status Experimental Control Protein Purification/Fractionation 1D PAGE 2D PAGE LC IEF Affinity Collect the Specific Cells or Tissue etc.. Isotopic Labeling ICAT/SILAC/PhIAT Extract the (sub)proteome of Interest Peptide Digestion Digestion Digestion Peptide Purification/Fractionation LC (SCX/RP) IMAC/TiO2 LC IEF Affinity Isotopic Labeling itraq/ 18 O. MS Anal. Qualitative LC-MS/MS Isotope Assisted Quantitative LC-MS/MS Labeling Free Quantitative LC-MS/MS Data Anal. MS Signal Processing Peptide/Protein Identification Peptide/Protein Quantitation Result Interpretation 4
5 Workflow of a Typical Proteomic Experiment Bottom-up approach Data-Dependent Acquisition (DDA) Nat Biotechnology, 2010 July;28(7):
6 Quantitative Approaches in LC-MS Based Proteomics MS-based Quantification ICAT, SILAC, dimethyl, etc Label-Free Journal of Proteome Research, 2008, 7, 51 61
7 Quantitative Approaches in LC-MS Based Proteomics MS/MS-based Quantification itraq, TMT, etc
8 MS-Based Proteomics Software Identification Software Quantification Software Other Software Mass Spectrometric Data Interpretation Statistics Software PTMs ID Software Analysis Software from MS Vendor ftware
9 Why Should Develop Universal MS Platform Vendor s Analysis Software Only support their MS raw data Analysis function is limited Their algorithm is not published License is expensive Universal MS Software Unlimited Unlimited Published Most software is free
10 Common MS-Based Proteomics Software Identification Software SEQUEST, Mascot, X!Tandem, Quantification Software ASAPRatio, Libra, Mascot,. Other Software Mass Spectrometric Data Interpretation MZmine, mmass, Statistics Software TPP, Scaffold, Analysis Software from MS Vendor ProteinLynx(Waters), ProteinDiscovery(Thermo),
11 Data Workflow for General MS-Based Proteomics 6300 Series Ion Trap LC/MS amazon X microtof-q II LCT-premire XE 6500 Series Accurate (Q-TOF) LC/MS 6400 Series Triple Quadrupole LC/MS? Triple quadrupole solarixtm Maxis Xevo TQ MS Raw Data Loading??? Quantitation ASAPRatio, Libra, etc.. MS/MS Peak List Extraction Peptide Identification MASCOT SEQUEST etc. Peptide Validation Peptide Prophet etc. Protein Validation Protein Prophet etc. Result Interpretation GO, DAVID, etc. NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 11 NOVEMBER 2004
12 The Common Data Format for the MS Data: mzxml
13 Proteomics Data Workflow for General MS-Based Proteomics MS Raw Data amazon X microtof-q II LCT-premire XE Converter for Different Instrumentations Triple quadrupole solarixtm Maxis Xevo TQ 6300 Series Ion Trap LC/MS mzxml / mzml Data Quantitation ASAPRatio, Libra, etc Series Triple 6500 Series Accurate Quadrupole LC/MS (Q-TOF) LC/MS MS/MS Spectra Extraction Peptide Identification MASCOT SEQUEST etc. Peptide Validation Peptide Prophet etc. Protein Validation Protein Prophet etc. Result Interpretation GO, DAVID, etc. NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 11 NOVEMBER 2004
14 Some of the MS Type Must Preprocessed Before Performing Proteomics Data Analysis Low Resolution Centroid MS Spectrum mzxml Data Analysis
15 Some of the MS Type Must Preprocessed Before Performing Proteomics Data Analysis MS Type 1 MS Type 2 MS Type 3 mzxml Data Analysis
16 The Common Data Format for the MS Data: mzxml MS Type 1 MS Type 2 MS Type 3 mzxml mzxml mzxml Selection of Correct Signal Processing Method Processed mzxml Data Analysis Noise Removal Deconvolution Mass Calibration
17 Proteomics Data Workflow for General MS-Based Proteomics Anal. Chem., 2013, 85 (2), pp
18 Proteomics Data Workflow for General MS-Based Proteomics Anal. Chem., 2013, 85 (2), pp
19 Proteomics Data Workflow for General MS-Based Proteomics MS Raw Data to XML Format Conversion mzxml/mzml Processing UniQua Quantitation ASAPRatio, Libra, etc.. MS/MS Peak Lists Extraction Peptide Identification MASCOT SEQUEST etc. Peptide Validation Peptide Prophet etc. Protein Validation Protein Prophet etc. Result Interpretation GO, DAVID, etc.
20
21 Implementation MS Data Processing mzxml Format Conversion MS Data Processing Software MS Data Analysis Mascot (Identification, Quantification) Output Interpretation
22 Implementation MS Data Processing mzxml/mzml Format Conversion MS Data Processing Software MS Data Analysis Mascot (Identification, Quantification) Output Interpretation
23 mzxml Format Conversion e=formats:mzxml Converter Download:
24 mzxml Format Conversion Download and Install ProteoWizard :
25 mzxml Format Conversion Execute MSConvert :
26 mzxml Format Conversion
27 mzxml Format Conversion
28 Implementation MS Data Processing mzxml/mzml Format Conversion MS Data Processing Software MS Data Analysis Mascot (Identification, Quantification) Output Interpretation
29 UniQua : A Universal MS Data Processing Software
30 The System Diagram of UniQua
31 mzxml File Transfer via FTP Open site manager and create a new site
32 mzxml File Transfer via FTP Upload your mzxml file(s)
33 The Web Service Interface of UniQua Type your user name, password then login
34 The Web Service Interface of UniQua Setup data processing parameters
35 The Web Service Interface of UniQua Select.mzXML file(s)
36 The Web Service Interface of UniQua Select instrument type
37 The Web Service Interface of UniQua Select MS1 and MS2 spectra whether be processed
38 The Web Service Interface of UniQua Smoothing function: select a appropriate smoothing width
39 The Web Service Interface of UniQua Centroid function and select appropriate peak height and define baseline for peak recognition
40 The Web Service Interface of UniQua Deisotope function
41 The Web Service Interface of UniQua Remove noise by: 1.appropriate MS/MS Resolution : Maximum resolution threshold (instrument dependent) 2.Peak Point: Minimum peak data point number threshold (instrument dependent) 3.Absolute Intensity Cutoff: A positive integer 4.Relative Intensity Cutoff : A positive integer(0 to 100)
42 The Web Service Interface of UniQua MS1 spectra calibration function (by one of the three calibration options)
43 The Web Service Interface of UniQua Check the precursor m/z by precursor residue in MS2 spectra
44 The Web Service Interface of UniQua Calibrate precursor m/z (by one of the three calibration options)
45 The Web Service Interface of UniQua Select a appropriate calibration method: 1. Using reference mass (setup reference m/z, tolerance and retention time range)
46 The Web Service Interface of UniQua Select a appropriate calibration method: 2. Using Lockspray (setup lockspray m/z and tolerance)
47 The Web Service Interface of UniQua Select a appropriate calibration method: 3. Using search result (setup m/z tolerance and retention time range), and the calibration file should be provided (example can be found on niqua/downloads.html )
48 The Web Service Interface of UniQua For MS2 level quantification
49 The Web Service Interface of UniQua Convert to MGF (for MASCOT search)
50 The Web Service Interface of UniQua Data processing progression Click Parameters : Show project parameters
51 The Web Service Interface of UniQua Click Logs : Show your project list. Projects are shown as: year/month/date/hour/min/sec.
52 The Web Service Interface of UniQua 1..ini file(s) is processing parameter 2. Example.mzXML is the original file 3. Example.out is data processing schedule 4. Example_p.mzXML is the processed file 5. Example_p.mgf was converted from Example_p.mzXML
53 Implementation MS Data Processing mzxml Format Conversion MS Data Processing Software MS Data Analysis Mascot (Identification, Quantification) Output Interpretation
54 Mascot : Protein Identification Software
55 Mascot : Protein Identification Software
56 Mascot : Protein Identification Software
57 Mascot : Protein Identification Software
58 Mascot : Protein Identification Software
59 Mascot : Protein Identification Software
60 Mascot : Protein Identification Software
61 Mascot : Protein Identification Software
62 Mascot : Protein Identification Software
63 Other Applications Software: SEQUEST and X!Tandem Search Software: Trans Proteomic Pipeline (TPP) MS-Level Quantification Metabolite Identification
64 To Contact Us Laboratory of MS-Based OMICS Research, Agricultural Biotechnology Research Center, Academia Sinica Principle Investigator: Dr. Yet-Ran Chen 陳 逸 然 博 士 Developer: Wei-Hung Chang 張 瑋 宏 TEL:
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