Protein Identification

Mass spectrometry in protein identification

Regardless of the question, the mainstay of proteomics is protein identification. In current laboratory practice, protein identification and mass spectrometry (MS) are nearly synonymous because MS allows for protein analysis from any sample of varying complexity, is high-throughput, and is quantitative. With MS, proteins can be identified at the intact (top-down) protein level or by using the more popular strategy, bottom-up proteomics. With the latter strategy, proteins are enzymatically digested down to their peptide components, then analyzed at the peptide level.

Thermo Scientific Orbitrap MS solutions enable both intact protein and peptide level workflows for in-depth and fast protein identification.

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MS techniques used for protein identification

Peptide sequencing by LC-MS/MS
Peptide sequencing by LC-MS/MS

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the primary workflow for most researchers when performing protein identification. Proteins are enzymatically digested to their peptide components, then analyzed by LC-MS/MS. The resulting sequence data are used to determine the original protein components of the sample. For single proteins, sequence usually confirms identity. When protein complexes are involved, this top-down proteomics approach determines the individual proteins that are part of the complex.

Top-down MS
Top-down mass spectrometry

Protein identification can be achieved without prior digestion at the intact protein level using gas phase fragmentation. This approach enables combinations of posttranslational modifications (PTMs) to be localized, permitting higher sequence coverage for individual proteins and characterization of proteoforms. Additional information, such as degradation products and sequence variants, can also be obtained within this type of experiment.