Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry.^[1][2] The major alternative workflow used in proteomics is called top-down proteomics where intact proteins are purified prior to digestion and/or fragmentation either within the mass spectrometer or by 2D electrophoresis.^[3] Essentially, bottom-up proteomics is a relatively simple and reliable means of determining the protein make-up of a given sample of cells, tissues, etc.^[4]

In bottom-up proteomics, the crude protein extract is enzymatically digested, followed by one or more dimensions of separation of the peptides by liquid chromatography coupled to mass spectrometry, a technique known as shotgun proteomics.^[5][6] By comparing the masses of the proteolytic peptides or their tandem mass spectra with those predicted from a sequence database or annotated peptide spectral in a peptide spectral library, peptides can be identified and multiple peptide identifications assembled into a protein identification.

Advantages

For high throughput bottom-up methods, there is better front-end separation of peptides compared with proteins and higher sensitivity than the (non-gel) top-down methods.^[7]

Disadvantages

There is limited protein sequence coverage by identified peptides, loss of labile PTMs, and ambiguity of the origin for redundant peptide sequences.^[7] Recently the combination of bottom-up and top-down proteomics, so called middle-down proteomics, is receiving a lot of attention as this approach not only can be applied to the analysis of large protein fragments but also avoids redundant peptide sequences.^[8]