title: Novel staining-free proteomic method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers.
abstract: A new proteomic staining-free method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI 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 pI markers extracted form the same piece excised from the gel. In this method, the pI markers are mixed with a protein mixture (a commercial malted barley protein extract) deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of focusing process. Several separated bands of the pI markers (including separated proteins) were excised and the pI 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 pI 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.
authors.affiliation: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
authors.forename: Josef
authors.initials: J
authors.lastname: Chmelík
authors.forename: Karel
authors.initials: K
authors.lastname: Mazanec
authors.forename: Karel
authors.initials: K
authors.lastname: Slais
mesh_terms: D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics
chemical_list: D011506:Proteins