Main steps during sample preparation are as follows:
Proteins are extracted from samples like tissues, cell cultures, serum etc., and analysed by mass spectrometry to identify post-translational modifications, to study protein complexes and protein-protein interactions. For ease of extraction, detergents are added in extraction and purification buffers. Most detergents are not compatible with mass spectrometric analysis, hence extra wash steps using PBS or HEPES or Tris. HCl is included (10-20 volumes) and elution of proteins with 0.1 M Glycine pH 2.5 is preferred to avoid detergent contamination.
Desalting of samples from chromatographic fractions were carried out using RP HPLC. RP HPLC samples contain Acetonitrile and TFA (low pH) so to neutralize the pH, Ammonium bicarbonate (1M) is added to the samples. Reduction of Disulphide bonds in the protein samples is achieved by adding 10mM DTT or 5mM TCEP and incubation of the samples for 10-30 min at 37 ℃- 50 ℃. DTT work well in the pH range 7-9, whereas TCEP works at wide pH range (2-11). Alkylation of cysteine residues is carried out by adding Iodoacetamide at a concentration of 10mM - 50mM and incubating the samples for 10min at 37℃. Following alkylation, an excess alkylating agent is removeby adding 40 mM DTT.
Protein mixtures are separated by SDS PAGE gels by 1D (based on molecular weight) or 2D (proteins are separated based on both isoelectric point and molecular weight) gel or separated as native complex by native PAGE. Then the gel is stained with gel stains compatible with Mass spec analysis. Gels can either be stained with Coomassie blue stain Colloidal Coomassie Blue-Stain (sensitivity 10-100 ng) or with glutaraldehyde free silver stain FOCUS-FAST Silver (sensitivity <1 ng) or zinc stain Reversible-Zinc-Stain (0.1-0.5 ng). The desired protein band is then excised from the gel using gel excision tools. The gel is then destained with compatible destaining solution DeStain/Blue-OUT followed by several washes with water to remove excess solutions. Then the gels were dehydrated by adding 100% acetonitrile. DTT was added to the dehydrated gels that convert cystines disulfide bonds into cysteine-free sulphydryl groups. These free sulphydryl groups are then converted to S- carboxyamido methyl-cysteine after adding alkylating agent Iodoacetamide. This will prevent proteins from reoxidizing.
Proteins are digested into small peptides by chemical methods or enzymatic methods. Chemicals like (CN)Br / 70% Formic acid are used. (CN)Br cleaves at C-terminal end of Methionine residues resulting in a peptide mixture of C-terminal Homoserine or Homoserine lactone. Enzymatic digestion is carried out by adding either Trypsin, Chymotrypsin, Lys-C, Asp-N or combination. Digestion of protein samples with Trypsin is widely used. Complete digestion of protein samples (10 µg) is achieved by incubating at 37 ℃ with trypsin to protein ratio of 1:20 for 4 hr with gentle agitation. Adding more trypsin results in trypsin autolysis and additional peaks are observed during mass spec analysis. Trypsin digestion is enhanced by adding LysC prior to trypsin digestion for some protein samples. Digestion is stopped by adding 5 µl of 1% TFA ( pH 2-3).
Gel enzyme solution is added to digest proteins within the gel piece and incubated on ice for 1 hour. Then the reaction is stopped by adding 25 mM Sodium bicarbonate solution. The solution is left overnight so that the peptides are extracted into solution. The peptides remained in the gel are further extracted by adding fresh gel solution or by adding 5 % Formic acid followed by 100 % Acetonitrile and incubated for 20-30 min. The peptide supernatants are pooled and vacuum dried. Peptides are then desalted and cleaned further before proceeding to mass spec analysis.
Low abundant proteins or peptides were purified and enriched before analysis. Phosphorylated proteins can be enriched by using IMAC beads or Titanium dioxide beads or both. The sample to be enriched should be passed in excess to the column and then washed with 10 column volumes of buffer and then eluted as a concentrated peptide solution using high pH elution buffer. By simply altering the pH of elution buffer mono (low pH with 1% TFA) and multi-phosphorylated peptides (high pH-11) can separately be collected as peptide enriched solutions.