title: A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea.
abstract: Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038, USA.
authors.forename: Sung Chan
authors.initials: SC
authors.lastname: Kim
authors.forename: Yue
authors.initials: Y
authors.lastname: Chen
authors.forename: Shama
authors.initials: S
authors.lastname: Mirza
authors.forename: Yingda
authors.initials: Y
authors.lastname: Xu
authors.forename: Jaeick
authors.initials: J
authors.lastname: Lee
authors.forename: Pingsheng
authors.initials: P
authors.lastname: Liu
authors.forename: Yingming
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000595:Amino Acid Sequence; D002457:Cell Extracts / Q000737:chemistry*; D011232:Chemical Precipitation; D019295:Computational Biology; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D014238:Trichloroacetic Acid; D014357:Trypsin
chemical_list: D002457:Cell Extracts; D011506:Proteins; D014238:Trichloroacetic Acid; D014357:Trypsin