LC/MS/MS Protein analysis

 The most common reason for analysis failure is high contamination levels by keratin. Keratin is a ubiquitous hair and skin protein present on dust, equipment and lab benches. Using high grade reagents (most notably SDS), cleaning all equipment prior to use and staining, and wearing gloves at all time will help minimize contamination.

 The choice of protein stain greatly influence the success of the MS analysis. We strongly recommend the use of Coomassie Blue staining as it is fully compatible with the analysis protocols. For more sentitive staining, one may use fluorescent dyes (eg. sypro, ruby). Typically a visible comassie stained band will contain enough material for a reliable analysis. We recommend to avoid silver staining since it was shown in many instances to interfere with gel extraction.

Sequencing by Edman degradation

 Peptides & proteins with blocked N-terminal amino groups (e.g. acetylated, formylated) will not sequence by Edman degradation.

 High molecular weight proteins (over 75 kDa) may prove difficult to be analyzed. In our experience, the foremost reason is sample quantity and / or purity. However, reasonably pure protein bands and comfortable amounts will allow for unambiguous sequence calls. Please contact us before sending your sample.

 Unmodified Cys and glycosylated residues will give a blank cycle (i.e. residue is cleaved off the peptide chain but is not detected by HPLC at 270 nm).

 Sample quality is critical. Any impurity that interfers with Edman’s chemistry (mainly amine-containing chemicals) will lead to poor results.