Orbitrap Illustration

Bottom-Up Proteomics

While more complex experiments such as relative and absolute quantification of proteins have become increasingly common, bottom-up protein identification continues to be a mainstay of proteomics. Its principal objective is to identify as many of the protein components of a biological sample as possible. Following digestion, peptides are identified by LC/MS. The resulting sequence data is used to determine the original protein components of the sample. With advances in mass resolution, mass accuracy, fragmentation technology and speed, bottom-up analysis can identify more proteins in more-complex samples than ever. Moreover, the techniques and principles involved are generally foundational for more complex experiments. Here we describe the basic bottom-up experiment, with attention to performance optimization for various sample types.

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Workflow Overview for Bottom-Up Proteomics

Bottom-up proteomics serves as the basis for much of the protein research undertaken in mass spectrometry laboratories today. The term ”bottom-up” implies that information about the constituent proteins of a biological sample are reconstructed from individually identified fragment peptides. To facilitate bottom-up MS analysis, proteins are subjected to proteolytic digestion, typically using trypsin. The resulting peptides are usually separated using one or more dimensions of liquid chromatography. The LC eluent is interfaced to a mass spectrometer using electrospray ionization and the fragment peptides are analyzed by MS.



Literature Highlight

Advancing cell biology through Proteomics in Space and Time (PROSPECTS)

Lamond AI, Uhlen M, et al.
Mol Cell Proteomics. 2012 Mar;11(3):O112.017731.

Pushing the Limits of Bottom-Up Proteomics with State-Of-The-Art Capillary UHPLC and Orbitrap Mass Spectrometry for Reproducible Quantitation of Proteomes

Lopez-Ferrer D, Blank M , Meding, et al.
Application Note 639