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0.5ml microcentrifuge tubes prewashed twice with 50% ACN/0.1% TFA.ZipTip® scx pipette tips (Millipore Cat.# ZTSCXS096) or ZipTip® C18 pipette tips (Millipore Cat.# ZTC18S096).SimplyBlue™ SafeStain (Invitrogen Cat.# LC6060).Trypsin Gold, Mass Spectrometry Grade (Cat.# V5280) and protocol.For a more streamlined protocol, see the protocol for In-Gel Digestion of Proteins Using Trypsin and ProteaseMAX™ Surfactant, Trypsin Enhancer in the ProteaseMAX™ Surfactant, Trypsin Enhancer, Technical Bulletin #TB373. The following procedure has been used successfully by Promega scientists. Numerous protocols for in-gel protein digestion exist (Flannery et al., 1989 Shevchenko et al., 1996 Rosenfeld et al., 1992). Trypsin is highly active and tolerant of many additives. The C-terminal arginine and lysine peptides are charged, making them detectable by MS. It is highly specific, cutting at the carboxyl side of arginine and lysine residues. It cleaves proteins into peptides with an average size of 700-1500 daltons, which is in the ideal range for MS (Laskay et al., 2013). Trypsin has become the gold standard for protein digestion to peptides for shotgun proteomics.
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Identification of peptides and subsequently proteins is completed by matching peptide fragment ion spectra to theoretical spectra generated from protein databases. The resulting complex mixture of peptides is analyzed by reverse-phase liquid chromatography (RP-LC) coupled to tandem mass spectrometry (MS/MS). Bottom-up proteomics focuses on the analysis of protein mixtures after enzymatic digestion of the proteins into peptides. These applications primarily employ bottom-up proteomics, where proteins of interest are digested with an enzyme such as trypsin and the resulting peptides are analyzed by mass spectrometry. Mass spectrometry is used for protein identification, the study of protein:protein interactions, characterization of post-translational modifications (e.g., phosphorylation, glycosylation, methylation and acetylation) and protein quantification (relative and absolute). Mass spectrometry is a leading analytical method in proteomics (Mann et al., 2001).