Proteomics measures proteins — the effector layer of life, which the transcriptome predicts only imperfectly. By mass spectrometry, thousands of proteins in a sample are identified and quantified, modifications are detected, a tumour is compared to its healthy tissue. Where RNA indicates what could be produced, proteomics measures what is actually present.
Principle and workflow
The dominant approach is called bottom-up (or shotgun): proteins are digested by a protease (most often trypsin) into peptides, separated by liquid chromatography — often at nano-flow — then ionised and fragmented in tandem mass spectrometry (MS/MS). Algorithms match the acquired spectra against a sequence database to identify peptides and infer proteins, under the control of a false-discovery rate (FDR, typically 1% via a target-decoy strategy). Quantification is done label-free or by isotopic labelling (TMT, SILAC) to compare conditions, or in a targeted manner (MRM/PRM) to precisely track a chosen panel of proteins.
Variants and options
A distinction is drawn between discovery (shotgun, broad and unbiased) and targeted proteomics (MRM/PRM, sensitive and quantitative on defined targets), and between label-free and labelling (TMT/iTRAQ, SILAC). Proteogenomics enriches the sequence database with the genomic and transcriptomic data of the same sample, to identify variants absent from public repositories. Added to these are specialties: phosphoproteomics and mapping of post-translational modifications, immunopeptidomics, interactomics by affinity purification (AP-MS), and the DDA or DIA acquisition modes.
When and why these techniques
Proteomics is chosen when the question concerns the proteins themselves: their expression level (which correlates only imperfectly with that of mRNAs), their modifications, the identification of sequence variants, or a differential comparison between tumour and healthy tissue. It complements transcriptomics without replacing it — pathways enriched at the protein level frequently do not appear in the RNA data.
Several limits weigh on interpretation. Dynamic range is the major challenge: highly abundant proteins mask the rarer ones, an extreme phenomenon in plasma. Proteome coverage remains incomplete, with missing values from one sample to another; inference of proteins from peptides is sometimes ambiguous (peptides shared between several proteins); bottom-up poorly identifies splice variants and does not exhaustively map post-translational modifications. The identification of sequence variants requires an enriched database, failing which the real forms escape generic repositories. Finally, hydrophobic or membrane proteins, those of very high molecular weight or highly variable (such as PfEMP1) remain technically demanding, and label-free quantification requires careful normalisation.
Inovarion’s expertise
Inovarion uses mass spectrometry to answer protein questions, in oncology as in infectious diseases. Several illustrations have been published: the integration of proteomics, genomics and transcriptomics refined the molecular classification of bladder cancer and revealed a therapeutic sensitivity — TRAIL-induced apoptosis in FGFR3-mutated tumours — that the transcriptome alone did not capture; a proteogenomic approach based on an enriched sequence database made it possible to reconstruct variants of the PfEMP1 protein that are hard to identify in severe malaria; and differential mass spectrometry, supplemented by a targeted MRM, quantified the exocytosis proteins in phaeochromocytoma. Discovery or targeting, labelling or label-free, sample preparation: everything depends on the question at hand.
See also: “Bulk” RNA-seq & differential transcriptomics ; Co-immunoprecipitation & protein interactions ; Bioinformatics.
Key publications
- Groeneveld et al. Proteogenomic Characterization of Bladder Cancer Reveals Sensitivity to Apoptosis Induced by Tumor Necrosis Factor-related Apoptosis-inducing Ligand in FGFR3-mutated Tumors. European Urology, 2024. Record → · PubMed
- Kamaliddin et al. From genomic to LC-MS/MS evidence: Analysis of PfEMP1 in Benin malaria cases. PLoS One, 2019. PubMed
- Houy et al. Dysfunction of calcium-regulated exocytosis at a single-cell level causes catecholamine hypersecretion in patients with pheochromocytoma. Cancer Letters, 2022. Record → · PubMed