title: Preparation of protein samples for mass spectrometry and N-terminal sequencing.
abstract: The preparation of protein samples for mass spectrometry and N-terminal sequencing is a key step in successfully identifying proteins. Mass spectrometry is a very sensitive technique, and as such, samples must be prepared carefully since they can be subject to contamination of the sample (e.g., due to incomplete subcellular fractionation or purification of a multiprotein complex), overwhelming of the sample by highly abundant proteins, and contamination from skin or hair (keratin can be a very common hit). One goal of sample preparation for mass spec is to reduce the complexity of the sample - in the example presented here, mitochondria are purified, solubilized, and fractionated by sucrose density gradient sedimentation prior to preparative 1D SDS-PAGE. It is important to verify the purity and integrity of the sample so that you can have confidence in the hits obtained. More protein is needed for N-terminal sequencing and ideally it should be purified to a single band when run on an SDS-polyacrylamide gel. The example presented here involves stably expressing a tagged protein in HEK293 cells and then isolating the protein by affinity purification and SDS-PAGE.
authors.affiliation: Ear Institute, University College London, London, United Kingdom. Electronic address: gary638@gmail.com.
authors.forename: Gary
authors.initials: G
authors.lastname: Glenn
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein / Q000379:methods; D012996:Solutions
chemical_list: D011506:Proteins; D012996:Solutions
keywords: Mitochondria purification; N-terminal sequencing; PVDF membrane; Preparation of protein samples; SDS-PAGE; Sucrose density gradient sedimentation; Whole cell lysates preparation