Computational protocol: Region of Interest analysis using mass spectrometry imaging of mitochondrial and sarcomeric proteins in acute cardiac infarction tissue

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Protocol publication

[…] MS/MS data were collected using a MALDI-QIT-TOF MS (AXIMA Resonance and MALDI-7090TM; Shimadzu). Spectra were exported to the Mascot search engine (Matrix Science, Boston, MA) using the following search parameters: taxonomy = Homo sapiens; database = SwissProt; MS tolerance = 0.2 Da; MS/MS tolerance = 0.3 Da; enzyme = trypsin; missed cleavage = 1. Peptides and proteins were identified using the Paragon algorithm provided with ProteinPilot 4.5 Beta (AB SCIEX, Danaher Corporation, Washington, D.C.) combined with the UniProt-Swiss-Prot database (version 2010-6, Homo sapiens) and sequences of the known contaminants (AB SCIEX). Matches were assigned with a significance threshold of P < 0.05. We performed false discovery rate (FDR) analysis after using the Proteomic System Performance Evaluation Pipeline (PSPEP) software (Danaher) for peptide identification. We performed label-free quantification of peptides using Protein Quantitation 1.0 MicroApp (PQMA), which is part of Peak View 1.1.1 (Danaher). We analysed liquid chromatography-tandem mass spectrometry (LC-MS/MS) data sets for all samples using Protein Pilot 4.5 beta. Subsequently, to construct a list of peaks for all detected unique peptides, the file was imported to the Peak View 1.1.1 platform using PQMA. Protein abundance was obtained using Marker View 1.2.1. Peptides with a confidence >0.95 were selected for export. Shared peptides, including contaminating peptides, were not exported. Proteins identified based on only one peptide were excluded. Each value was normalized to the sum of the total area of individual sample. The reliability of the identification was validated by repeating the post-separation software analysis. […]

Pipeline specifications

Software tools Mascot Server, ProteinPilot
Databases UniProt
Application MS-based untargeted proteomics
Diseases Myocardial Infarction
Chemicals Adenosine Triphosphate