What is Proteomics?
Proteomics is the large-scale study of proteins—their structures, functions, and interactions. Unlike the relatively static genome, the proteome is dynamic, varying across tissues, conditions, and time.
Mass Spectrometry Basics
The Workflow
Sample preparation: Protein extraction and digestion (trypsin)Separation: Liquid chromatography (LC) separates peptidesIonization: Electrospray ionization (ESI) creates charged peptidesMass analysis: Measures mass-to-charge (m/z) ratiosFragmentation: MS/MS fragments peptides for identificationTypes of Acquisition
DDA (Data-Dependent Acquisition): Sequences most abundant ionsDIA (Data-Independent Acquisition): Fragments all ions in windowsTargeted (SRM/MRM): Monitors specific peptidesComputational Analysis
Database Searching
Tools like MaxQuant, Mascot, or MSFragger match spectra to peptide databases:
Calculate theoretical spectra from protein sequencesScore observed vs. theoretical matchesApply FDR control (typically 1%)Quantification Methods
Label-free: Compare ion intensities across runsTMT/iTRAQ: Chemical labels for multiplexingSILAC: Metabolic labeling with heavy isotopesApplications
Biomarker discovery: Identifying disease signaturesDrug target validation: Confirming target engagementPTM analysis: Mapping phosphorylation, glycosylationInteractomics: Mapping protein-protein interactionsBest Practices
Always include biological and technical replicatesNormalize appropriately for your quantification methodValidate key findings with orthogonal methods (Western blot, ELISA)
