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.

Generating Peptide Titration-Type Curves Using Polymeric Reverse Micelles As Selective Extraction Agents along with Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Detection

“…Before using these polymeric materials to separate peptide mixtures, however, we first test the performance of these materials on compounds with well-defined p K a values: a series of fluorescent p I markers. Such markers are commonly used as reference compounds in isoelectric focusing. , Initially studying these molecules is useful because these compounds also allow us to quantify the extraction via fluorescence spectroscopy. Analysis of these molecules by fluorescence is needed because MALDI-MS of peptides is semiquantitative at best.…”

Robustness and accuracy of high speed LC–MS separations for global peptide quantitation and biomarker discovery☆

“…For traditional off-line peptide fractionation, peptide IEF has several advantages over other methods. It allows the inclusion of fluorescent isoelectric point (pI)-markers to aid matching fractions across experiments [38,39] and adds the experimental peptide pI information of each identified peptide. Indeed Cargile and co-workers have shown the utility of peptide pI in combination with accurate mass alone and tandem MS-based approaches [40].…”

pI Determination of Native Proteins In Biological Samples

“…In the past couple of decades, several advances have been made in the IEF protocol to achieve specific objectives. For instance, optimization of the IEF procedure has been utilized to (1) detect particular types of proteins (e.g., Selenoproteins) (Sonet, Mounicou, & Chavatte, 2018), (2) employ specific ions (e.g., the euphorium, Eu 3+ ) in the pI determination of proteins (Pihlasalo, Auranen, Hanninen, & Harma, 2012), (3) facilitate mass spectrometry-based identification of proteins (Chmelik, Mazanec, & Slais, 2007), and (4) enable peptide-based pI calculations of phosphorylated or acetylated proteins (Gauci, van Breukelen, Lemeer, Krijgsveld, & Heck, 2008), and so on.…”