title: A Robust Protocol for Protein Extraction and Digestion.
abstract: Proteins play a key role in all aspects of cellular homeostasis. Proteomics, the large-scale study of proteins, provides in-depth data on protein properties, including abundances and post-translational modification states, and as such provides a rich avenue for the investigation of biological and disease processes. While proteomic tools such as mass spectrometry have enabled exquisitely sensitive sample analysis, sample preparation remains a critical unstandardized variable that can have a significant impact on downstream data readouts. Consistency in sample preparation and handling is therefore paramount in the collection and analysis of proteomic data.Here we describe methods for performing protein extraction from cell culture or tissues, digesting the isolated protein into peptides via in-solution enzymatic digest, and peptide cleanup with final preparations for analysis via liquid chromatography-mass spectrometry. These protocols have been optimized and standardized for maximum consistency and maintenance of sample integrity.
authors.affiliation: Canary Center at Stanford for Cancer Early Detection, Stanford University, 3155 Porter Drive, Palo Alto, CA, 94304, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Atallah
authors.affiliation: Canary Center at Stanford for Cancer Early Detection, Stanford University, 3155 Porter Drive, Palo Alto, CA, 94304, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Flory
authors.affiliation: Canary Center at Stanford for Cancer Early Detection, Stanford University, 3155 Porter Drive, Palo Alto, CA, 94304, USA. paragm@stanford.edu.
authors.forename: Parag
authors.initials: P
authors.lastname: Mallick
mesh_terms: D002853:Chromatography, Liquid; D006868:Hydrolysis; D066298:In Vitro Techniques; D010447:Peptide Hydrolases; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D011506:Proteins; D010447:Peptide Hydrolases
keywords: Acetone precipitation; Enzymatic solution digest; Liquid chromatography-mass spectrometry; Protein extraction; Proteomics