Protein Digestion

For bottom-up proteomics proteins are digested into smaller, easier to handle peptides, which are then separated by on-line HPLC and analyzed by the mass spectrometer (LC-MS). It is critical to avoid/minimize any contaminations during your entire sample preparation that could interfere with the subsequent LC-MS analysis.
Salts, detergents and plasticisers are ionizable contaminations that cause major problems during the LC-MS analysis. They compete with the peptides binding to the column and detection by the mass spectrometer and thus will drastically decrease the overall sensitivity of the analysis.
Complex biological matrices may not only be rich in proteins but also rich in metabolites, lipids, nucleic acids, sugars, and other molecules. If not removed, they will also compete with the peptides for analysis.
Therefore, the primary goal should be to eliminate contamination and to increase the amount/number of peptides in a sample. If at all possible, systematic preventive elimination of contaminants is preferable over retrospective reduction of contaminants.
Equally important is an effective digestion protocol that produces few missed cleavages, few unspecific cleavages, and few undesired side reactions. If a peptide is present in properly cleaved,a missed cleaved and/or modified form, its signal intensity will be distributed into the number of forms present, decreasing its signal intensity and increasing the sample complexity (i.e. the number of detectable peptide ions). For example use of urea solution can lead to carbamylations (via its decomposition to ammonium cyanate) when using aged solutions or when used at elevated temperatures (above 25oC)

Avoid detergents that are not mass spec compatible, including:

  • NP-40
  • TritonX (any derivative)
  • Igepal (any derivative)
  • Brij-35 (or any derivative)
  • Tween-20
  • OTG
  • SDS
Dilution, washing, and detergent removal columns often do NOT remove enough detergent for successful analysis of your sample, and can lead to massive contamination of the mass spectrometer and HPLC/column.
In addition detergent removal also leads to sample loss!

Use "mass spec friendly" detergents to keep the proteins in solution:

Starting out with clean HPLC grade solvents and keeping them clean is very important.
Avoid Contaminations (pdf)

Enzymatic Digestion Protocols

List of enzymes and specificity Expasy Peptide Cutter Page

Jimmy's UWPR Protein digestion calculator

List of digestion protocols

Digestion Enzymes

Chemical cleavage

  • CNBr: Cyanogen bromide hydrolyzes peptide bonds at the C-terminus of methionine residues converting Met to Homoserine
  • BNPS-Skatole: BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole] is a mild oxidant and brominating reagent that cleaves at the C-terminus of tryptophan
  • Formic Acid: Cleaves at the C-terminus of Asp
  • Hydroxylamine (NH2OH): Cleaves at the C-term. of Asn and at the N-term. of Gly
  • Iodosobenzoic acid: Cleaves at the C-terminus of Trp
  • NTCB +Ni (2-nitro-5-thiocyanobenzoic acid ): Cleaves at the N-terminus of Cys

Isotopic labeling
There are various chemical labeling strategies for concurrent peptide identification and multiplexed proteomics quantitation by mass spectrometry. Most quantitative proteomics reagents incorporate stable isotopes into the isobaric tag portion of the reagents and are used to label at the protein or peptide level. Check out their protocols, most start with the digestion followed by labeling:

  • Isotopic Labeling Overview UWPR page

  • Sigma iTRAQ (Isobaric tags for Relative and Absolute Quantification) page
    Amine-reactive, 8-plex reagents

  • ThermoPierce TMT Overview (Tandem Mass Tag) Reagents page
    Amine-reactive, 6-plex Tandem Mass Tag Reagents page
    Amine-reactive, 10-plex Tandem Mass Tag Reagents page
    Cysteine-Reactive, 6-plex Tandem Mass Tag Reagents page
    Carbonyl-reactive, 6-plex aminoxyTMT Reagents page

  • Planet Orbitrap TMT Overview (Tandem Mass Tag) Reagents page

List of peptide cleanup protocols for LC-MS