Novel staining-free proteomic method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers

✍ Scribed by Josef Chmelík; Karel Mazanec; Karel Šlais

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 492 KB
• Volume: 28
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.200600529

✦ Synopsis

Abstract

A new proteomic staining‐free method for simultaneous identification of proteins and determination of their p__I__ values by using low‐molecular‐mass p__I__ markers is described. It is based on separation of proteins in gels by IEF in combination with mass spectrometric analysis of both peptides derived by in‐gel digestion and low‐molecular‐mass p__I__ markers extracted form the same piece excised from the gel. In this method, the p__I__ markers are mixed with a protein mixture (a commercial malted barley protein extract) deposited on a gel and separated in a pH gradient. Color p__I__ markers enable supervision of progress of focusing process. Several separated bands of the p__I__ markers (including separated proteins) were excised and the p__I__ markers were eluted from each gel piece by water/ethanol and identified by MALDI‐TOF/TOF MS. The remaining carrier ampholytes were then washed out from gel pieces and proteins were in‐gel digested with trypsin or chymotrypsin. Obtained peptides were measured by MALDI‐TOF/TOF MS and proteins were identified via protein database search. This procedure allows omitting time‐consuming protein staining and destaining procedures, which shortens the analysis time. For comparison, other IEF gels were stained with CBB R 250 and proteins in the gel bands were identified. Similarity of the results confirmed that our approach can give information about the correct p__I__ values of particular proteins in complex samples at significantly shorter analysis times. This method can be very useful for identification of proteins and their post‐translational modifications in prefractioned samples, where post‐translational modifications (e.g., glycation) are frequent.