Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Abstract

Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.

Proteomic analysis of day–night variations in protein levels in the rat pineal gland

“…In-gel digestions were performed as described by Ross et al [22]. Briefly, the excised gel plugs were washed in digestion buffer (50 mM NH 4 HCO 3 , pH 7.8)/ACN (60:40) and dried by vacuum centrifugation.…”

Acid‐labile surfactant assists in‐solution digestion of proteins resistant to enzymatic attack

“…Acid‐labile surfactant (ALS) was introduced as a replacement for sodium dodecyl sulfate (SDS) in polyacrylamide gel electrophoresis (PAGE) in 1999, and the first experiments describing its use from testing laboratories around the world have appeared in print 1–4. ALS is a long‐chain derivative of 1,3‐dioxolane sodium propyloxy sulfate which degrades at low pH.…”

Deciphering tissue‐based proteome signatures revealed novel subtyping and prognostic markers for thymic epithelial tumors

“…After careful review of all the histological subtypes, lysis buffer was added into each sample, in which the corresponding tissue block was precut into small pieces (~ 1 mm 3 ). The lysis buffer contains 0.2% homemade acid‐labile surfactant (ALS) (Ross et al , ) in 20 m m HEPES buffer with 1X protease inhibitor (Roche, Basel, Switzerland) and was optimized specially for thymus tissue protein extraction in our previous study, resulting in a high recovery of versatile proteins (Sun et al , ). All the samples were placed individually in homogenization tube with precooled ceramic beads at 4 °C.…”