Why Scripts for Precision Proteomics of Hybrid Mixtures
While “1.0” focused on instrumentation, “Proteomics 2.0” — aka Precision Proteomics — is about application, particularly for translational clinical research. As such, the emphasis shifts from acquisition accuracy to dependable data analysis that is robust, sensitive, and reproducible. This means bioinformatics, the long misunderstood and neglected part of proteomics, becomes the critical success factor.
Unlike first generation tools reliant on statistical models for low-accuracy data, precision bioinformatics is necessarily a back-to-basics direct interpretation of raw mass/charge (m/z) measurements. This is a paradigm shift from over-reliance on inscrutable search scores and subjective probability models. It also requires flexible data-mining tools rather than simplistic push-button PC programs.
Is the bone broken? Best to look at the raw x-ray image, and not just some software summary.
Is the targeted low-abundance protein present? Best to directly compare measured vs. predicted m/z’s. Same principle.
Before search engines were invented, every mass spectrometry researcher understood correct identifications meant small delta-masses, which forms the foundation of SorcererScore(tm). Two decades later, many seem to have forgotten core basics and became over-dependent on complex formulas (search scores, probabilities, error rates) that are always imprecise and often misleading.
Here, we illustrate the precision bioinformatics paradigm, including how scripts provide flexibility to handle hybrid mixtures that canned software cannot.
In most cases, we expect to provide SORCERER GEMYNI sample scripts free of charge, which licensed users can customize. Simple scripts can take only a few hours or even minutes to write. These scripts can run on a low-priced cloud account (SORCERER Storm) up to the high-performance physical SORCERER Pro iDA system.
Why hybrid mixtures
Conventional workflows handle simple experiments involving uniformly processed, simple mixtures of known abundant proteins. This means proteins with known sequences from one sample are digested by one enzyme (typically trypsin) into peptides, which are dissociated within a tandem mass spectrometer by one mechanism (typically CID or HCD resulting in b-ions and y-ions).
Hybrid mixtures are important for advanced research for differential quantitation or improved peptide identification:
Heavy isotope-labeled peptides spiked into a bio-sample
CID vs. ETD spectra (b/y-ions vs. c/z-ions)
Light vs. heavy isotope labeling of specific amino acids in cells (SILAC)
Light vs. heavy isotope labeling of many amino acids in animals (SILAM)
Peptides from same protein mixture but digested with different enzymes, for sequencing unknown proteins