Methodology for MALDI-TOF MS Data Analysis - IIT...

Proteomics

Methodology for MALDI-TOF MS Data Analysis

Methodology for MALD-TOF Data Analysis

Once the peptide mass data of individual protein is obtained fromMALDI-TOF, the next step is for identification of protein throughdatabase search. The database matching for query sequencedepends on lot of parameters, which need to be optimized tocome up with the best fit.

Learning Objectives:

After interacting with this learning object, the learner will be able to:

• Operate on setting up the parameters.• Analyze the result output from the database.• Assess the troubleshooting steps involved in the experiments.

• Prepare in handling the database search tools.

Note: The current IDD exists in two modes- interactive and automatic.Students taking lab course should select interactive (set as default),while the automatic mode may be selected for general users.

In MS analysis, sample firing data that is generatedcan be saved in the excel format by making a note ofm/z of the peaks or by saving the spectrum in ASCIIformat.

Data Input


Open the matrixscience browser window to carry outonline data analysis. In case of peptide analyses,please select peptide mass fingerprint option. Moreinformation can be obtained by clicking each of thehighlighted buttons.

Parameter Settings


The details for each of the parameters must beentered depending on the background of sample typeand its source. Output data for the best hit depends alot on the input parameter details.

Parameter Settings


Provide details like the user id, which may be requiredin case of any network problem, so that the outputdata can be mailed to the user.

Parameter Settings


The following paramaters should be selected

Databases: The primary sequence protein databases,including NCBI and SwissProt against which the querywill run.

Enzyme: Used during sample preparation before itsmass spectrometric analysis.

Missed Cleavage Allowed: Occurrence of partial digestsduring trypsinolysis of sample protein at one or twoArginine and Lysine sites.

Parameter Settings


In case of Taxonomy: the search query must be limitedto a particular species or a group of species for whichthe sample belongs.

Parameter Settings


For Fixed Modifications: These are modifications thatare applied collectively across the database toaccount for change in mass of specific residue/s.

Variable Modifications: These are mass changessuspected to occur during sample handling andaccounted for by increasing the number of primarysequences compared against experimental masses.

Parameter Settings


The parameters can be changed depending uponuser needs.

Protein Mass: Is the mass of intact protein in theform of a contiguous stretch including allmatched peptides.

Mass Values: To specify the type of charge of theanalyte being examined.

Monoisotopic Mass Vs Average Mass Value:Depending upon the mass accuracy of aspectrometer, the experimental massescalculated for identification of analyte byPeptide mass fingerprinting either by choosingmonoisotopic mass or the average mass of itsisotopic elements.

Parameter Settings


A Query can be uploaded by feeding data or bybrowsing the file from the database. The result outputin the form of the hits can be selected and decoyhelps to search with same parameter across database.

Parameter Settings


Do make sure all the parameters are set and massdata is uploaded, user can click the start searchbutton. Depending on the set parameters the search inthe database begins.

Parameter Settings


The result output is folded into three sections:

In Section 1: the summary of set parameters definedby user.

Section 2: Mascot Score Histogram. The number ofprotein hits with score is plotted along the graph.

Section 3: Summary report in which matched proteinsfrom the database, with the details of importantparameters are displayed either in concise format,protein format and the data can be exported too.

Data Output


If the search parameters are not the best fit, thesoftware generates an error message. Depending onthe error message user needs to change the parametersetting and do the search again.

Data Analysis


In Section 2: Mascot Score Histogram, the number ofprotein hits and their score is displayed along thegraph.

Data Analysis


The section: 3 concise protein summary report givesout more details about the protein identification.Matched proteins from the database, with name,mass, score and other details of important parametersare given. For individual protein information, thedetails can be obtained by clicking on the blue link foreach protein.

Data Analysis


In protein view section, displays matching of the querypeptide to the protein sequence in the database. Thesequence type, the matched region, what is theexpected and calculated values of the query peptideand sequence details.

Data Analysis


In protein summary report, index displays the veryconcise details followed by the details of the each ofthe hits separately. The details contain the ID, Mass,score, expected value and number of matches.

Data Analysis


In export search result report, user has options fordata information to be stored with respect toparameters selected. The result can be exported andsaved, the data can be later taken for pathwayanalysis and literature study. In case user has doneMS/MS of a particular protein spot, the searchparameters are all the same with inclusion of fewmore parameters for the best fit.

Data Analysis


For better protein identification and to increaseprotein score, CID of each peak generated is carriedout to generate MS/MS data. For such data analysis,MS-fit option is selected from the matrix sciencebrowser window. In MS/MS search tool, more inputparameters like Quantitation, MS/MS tolerance,peptide charge, instrument etc. in addition to thefields for PMF and rest other parameters are similar tothat of Peptide mass fingerprint.

Data Analysis


Depending upon the process carried out for datageneration, a selection in the Quantitation must bemade. Quantitation of the extracted protein, peptide,mixture of both, extracted from the differentinstruments before the MS analysis. For example:iTRAQ, Tandem mass Tags for fixed mass/chargevalues, ICAT, ICPL for precursors within a single dataset, SILAC for ion fragments peaks, XICs for precursorsin multiple data set etc. For more information oniTRAQ, ICAT, ICPL and SILAC follow the respectiveIDDs.

Data Analysis


Define the instrument that has been used to generatethe raw data.

Data Analysis


All the parameters added must be relevant to thebackground of sample details to get the best hit fromthe database search.

Data Analysis


The result output generated is almost similar tomascot output. The Tandem MS protein analysis is usedto obtain protein identities from each of thesequenced peptides. The results page begins with alist of probable protein identities and their respectivesources. The score histogram provides details similarto the PMF analysis, with the probability distributionbeing displayed graphically. The green shaded regionis indicative of a match that has greater than 5%chance of being random while the red peak indicatesthat the chances of a random match is less than 5%.

Data Analysis


The summary report lists all the protein matchesobtained from the database search with theirrespective molecular weight, protein score, sourceorganism and details regarding each of its fragmentedpeptides. Further information about any of the proteinsequences can be obtained by clicking on thecorresponding protein link. Data regarding each of thepeptide fragmentation patterns can also be obtainedby clicking on the peptide link indicated by the querynumber.

Data Analysis


Regarding the protein score, molecular weight,isoelectric point, the sequence coverage of theprotein etc. Protein scores above 67 are consideredsignificant and greater the percentage sequencecoverage more is the number of matching peptides forthat particular protein. All sequences are displayedwith the matching sequences being indicated in red.

Data Analysis


The protein view obtained on selecting a particularprotein link, is very similar to the protein viewobserved in PMF. It provides details Information abouteach of the matched peptides is also displayed. Thestart and end amino acid positions, calculated andexperimental molecular weights, number of missedtryptic cleavages, sequence of each peptide fragmentand their corresponding ion scores are shown. Thehighest ion scores are used for computing the finalprotein score.

Data Analysis


Each peptide in Tandem MS/MS undergoes a secondround of fragmentation when it passes through thesecond mass analyzer before it reaches the detector.This provides significantly larger amount ofinformation regarding each peptide fragment whichcan be viewed by clicking on the peptide linksprovided in the summary report. The fragmentationpattern is displayed graphically, which can be zoomedinto as per the requirement by adjusting the x-axisplot values.

Data Analysis


At low collision energy, each peptide fragment iscleaved at the amide bond which can result in theformation of two types of ions – the y ion & b ion. In y-ions, the positive charge is retained on the C-terminusof the peptide ion while in b-ions; charge is retainedon the N-terminal. These ion masses can be used tocompute the amino acid sequence by calculating themass difference between consecutive ions. Each massdifference value corresponds to a particular aminoacid, which can be obtained from a standardinformation table. The y-ion series & the b-ion seriesrun opposite to each other as indicated in the exampleabove. Please go through the future IDDs for moreinformation and better understanding of the concept.

Data Analysis


Methodology for MALDI-TOF MS Data Analysis - IIT...

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