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MATRIXSCIENCE.COM
DOES PROTEIN FDR HAVE ANY MEANING? Does protein FDR have any meaning? Its easy to grasp the concept of using a target/decoy search to estimate peptide false discovery rate. You search against a decoy database where there are no true matches available, so the number of observed matches provides a good estimate of the number of false matches in the results from the target. MASS-TOLERANT VS ERROR TOLERANT The frequency distributions for the bins illustrated in Figure 2 of the paper are narrow Gaussians, but some of the other bins with high counts extend over a very wide mass range and are not well fitted by a Gaussian, so have been labelled artefact. INSTRUMENT SPECIFIC MS/MS ION SERIES MATCHING 2 Ion series in Mascot 1.7 •a, b, and y ions •Neutral loss of 17 from all ions •2+ ions if precursor was 2+ or higher Room for improvement? •More ion series, esp. high energy fragments •Internal fragments •Neutral loss of 17 or 18 is composition dependent In Mascot 1.7, all MS/MS data was matched and scored using the a, b, and y ion series. DATABASE SEARCHING FOR PROTEIN IDENTIFICATION AND 5 If the mass spectrum of your peptide digest mixture looks as good as this, and it is a single protein, and the protein sequence or something very similar is in the database, your chances PHOSPHORYLATION SITE ANALYSIS 3 ASMS 2004 Search for phosphorylation, May 13 2004 - 656 messages One of the best sources of information on phosphorylation is the ABRF email discussion group. SOURCE CODE FOR THE SHAPIRO-WILK W TEST Source code for the Shapiro-Wilk W test algorithm /* ##### # File: ms_shapiro_wilk.cpp # # Mascot Parser STEP AWAY FROM THE IODOACETAMIDE Step away from the iodoacetamide. In our July newsletter, we featured a paper from Torsten Müller and Dominic Winter, University of Bonn, concerning alkylation artefacts.Some of their findings were quite shocking. For example, differences of more than 9 fold in numbers of identified methionine-containing peptides for in-gel digested samples between iodine- and non-iodine-containing GETTING THE MOST OUT OF YOUR MASCOT SERVER HARDWARE A Mascot Server needs a minimum of 16GB of RAM on a standalone computer or the head node of a cluster. Less than 16GB may mean Mascot is not able to bring very large databases on line. For larger Mascot Server licenses, with 3 or more CPU’s, we recommend an additional 1GB of RAM per a core. If the Mascot Server has insufficient RAM the ION SERIES FOR ETHCD Ion series for EThcD. The ion series considered during a Mascot search are selected by choosing an instrument type. If you have Mascot Server in-house, you can edit existing instrument types and create new ones. There are 17 ion series available, and Mascot takes an iterative approach to scoring. If the number of matched peaks in a particular BACK TO BASICS 5: PEPTIDE-SPECTRUM MATCH STATISTICSSEE MORE ONMATRIXSCIENCE.COM
DOES PROTEIN FDR HAVE ANY MEANING? Does protein FDR have any meaning? Its easy to grasp the concept of using a target/decoy search to estimate peptide false discovery rate. You search against a decoy database where there are no true matches available, so the number of observed matches provides a good estimate of the number of false matches in the results from the target. MASS-TOLERANT VS ERROR TOLERANT The frequency distributions for the bins illustrated in Figure 2 of the paper are narrow Gaussians, but some of the other bins with high counts extend over a very wide mass range and are not well fitted by a Gaussian, so have been labelled artefact. INSTRUMENT SPECIFIC MS/MS ION SERIES MATCHING 2 Ion series in Mascot 1.7 •a, b, and y ions •Neutral loss of 17 from all ions •2+ ions if precursor was 2+ or higher Room for improvement? •More ion series, esp. high energy fragments •Internal fragments •Neutral loss of 17 or 18 is composition dependent In Mascot 1.7, all MS/MS data was matched and scored using the a, b, and y ion series. DATABASE SEARCHING FOR PROTEIN IDENTIFICATION AND 5 If the mass spectrum of your peptide digest mixture looks as good as this, and it is a single protein, and the protein sequence or something very similar is in the database, your chances PHOSPHORYLATION SITE ANALYSIS 3 ASMS 2004 Search for phosphorylation, May 13 2004 - 656 messages One of the best sources of information on phosphorylation is the ABRF email discussion group. SOURCE CODE FOR THE SHAPIRO-WILK W TEST Source code for the Shapiro-Wilk W test algorithm /* ##### # File: ms_shapiro_wilk.cpp # # Mascot Parser STEP AWAY FROM THE IODOACETAMIDE Step away from the iodoacetamide. In our July newsletter, we featured a paper from Torsten Müller and Dominic Winter, University of Bonn, concerning alkylation artefacts.Some of their findings were quite shocking. For example, differences of more than 9 fold in numbers of identified methionine-containing peptides for in-gel digested samples between iodine- and non-iodine-containing ION SERIES FOR ETHCD Ion series for EThcD. The ion series considered during a Mascot search are selected by choosing an instrument type. If you have Mascot Server in-house, you can edit existing instrument types and create new ones. There are 17 ion series available, and Mascot takes an iterative approach to scoring. If the number of matched peaks in a particular GETTING THE MOST OUT OF YOUR MASCOT SERVER HARDWARE A Mascot Server needs a minimum of 16GB of RAM on a standalone computer or the head node of a cluster. Less than 16GB may mean Mascot is not able to bring very large databases on line. For larger Mascot Server licenses, with 3 or more CPU’s, we recommend an additional 1GB of RAM per a core. If the Mascot Server has insufficient RAM the SOLVING A PUZZLE WITH MASCOT DISTILLER DE NOVO Under Peak profile, set all three width values to 0.02. Choose Process scan from the Processing menu or the toolbar. You should now have something similar to this: Click to view full size image. From the Tools menu, choose Preferences, Sequence tag / De Novo. Since we cannot assume this is a tryptic peptide, set Enzyme to Specific andnon-specific.
COMMON MYTHS ABOUT PROTEIN SCORES A paper might say the authors accepted “all proteins with score > 51″ or “protein scores greater than 67 (p* Home
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