Confident Protein ID using Spectrum Mill Software

Transcription

1 Welcome to our E-Seminar: Confident Protein ID using Spectrum Mill Software Slide 1

2 Spectrum Mill Informatics Software Start with batches of raw MS data! Sp ec t ru m Mi ll Biologist-friendly answers! Slide 2

3 Spectrum Mill: A Web Server Based Application With a Full Range of Tools Slide 3

4 Spectrum Mill MS Proteomics Workbench A collection of informatics tools to convert MS and MS/MS spectra into protein identities and abundances Extraction of quality data (not noise!) from raw data files Support for analyses from multiple instrument types in multiple data formats Rapid protein database search Easy review/validation of peptide results (including MS/MS spectra) Mass-based semiquantitative analysis for first-order expression-level comparisons and also ICAT support Flexible summary view for reviewing results from single and multiple experiments Powerful de novo sequencing for peptides not found in the database Slide 4

5 Typical Workflow File_1.d File_2.d File_3.d... File_n.d Raw spectra Create results file (*.mgf, *.dta) Database search High quality Medium Low quality quality Low quality Slide 5

6 Spectrum Mill Workflow File_1.d File_2.d File_3.d... File_n.d Raw spectra Preprocessed spectra Database search High quality Medium Low quality quality Low quality Slide 6

7 Spectrum Mill Workflow: An Iterative Approach to Reducing Complex Datasets Database search Preprocessed spectra Medium quality Low quality High quality Medium Low quality quality Low quality Autovalidation of the best Valid Valid Valid Manual validation Slide 7

8 Extractor Program Pulls Only the Quality Spectra From the Raw Data File Merges scans from same precursor within a specified time window Reduces the number of noise spectra extracted with minimum sequence tag length and S/N parameters Determines charge state where possible Uses centroided data which reduces data file size Slide 8

9 What is a Mass Tag? A mass tag or sequence tag is a continuous string of amino acids that can be created by following a path from high mass to low mass that links MS/MS ions separated by the mass of an amino acid MS/MS spectrum y 2 y 4 y 3 y 1 y 5 LKECCDKPLLEK y 5 y 4 y 3 y 2 y 1 Slide 9

10 MS/MS Search Parameters Select data set Select database and digest conditions Select search mode Slide 10

11 Autovalidation of the Top-Scoring Matches Slide 11

12 Peptide Summary for Rapid Review and Manual Validation Just a single click to review the mass spectrum Slide 12

13 Flexible Display of MS/MS Search Results Protein Summary View sorted by Molecular Weight LC/MS of 1D gel bands Slide 13

14 Protein Detail View Shows Related Proteins Slide 14

15 MS/MS Search Results: Details of a Protein Match Select %AA coverage to view details Slide 15

16 Direct Link to NCBI: Protein Information Slide 16

17 Protein-Peptide Distribution Mode for Examining 2D LC/MS/MS Data Each column = 1 salt step Slide 17

18 Overview Facilitates Cross-Experiment Results Review and Displays Relative Quantitation Quantitation based on precursor mass chromatograms Slide 18

19 ICAT Calculations on Both the Peptide and Protein Levels Peptide mode Protein mode Slide 19

20 De Novo Sequencing Result: Sherenga Report Slide 20

21 Monitoring the Progress of Data Processing 6252 raw MS/MS spectra in 7 files 1436 MS/MS spectra after extraction and filtering 554 MS/MS spectra interpreted and validated Slide 21

22 How Long Does Processing Take? 2D LC/MS/MS analysis of E. coli sample (8 salt steps) 12 hours 40 minutes of data acquisition 11,254 raw MS/MS spectra 1,644 prequalified MS/MS spectra 81 proteins identified and validated 8 minutes!! Slide 22

23 Spectrum Mill Client / Server PC Configurations Minimum Server Requirements: Hardware NIC (TCPIP protocol), 2GB RAM, 2.2GHz dual CPU, and minimum 120GB hard drive Recommended Server: Compaq EVO XW8000 Dual 2.8 GHz WorkStation (2x200GB HDs) configured by SHI Slide 23

24 Spectrum Mill MS Proteomics Workbench Comprehensive data review and management of complex MS proteomics data Compare/correlate results across multiple samples Store and review data from large projects Visualize data from either a biology or mass spectrometry perspective Comprehensive review of results from protein database search and de novo sequencing Open platform for processing data from multiple vendors and LC/MS techniques Commercial rights to Spectrum Mill (by Millennium Pharmaceuticals, Inc.) are exclusively licensed to Agilent Technologies. Slide 24