Mass spectrometric characterization of low‐molecular‐mass color pI markers and their use for direct determination of pI value of proteins

Abstract

The use of low-molecular-mass color pI markers for the determination of pI values of proteins in gel isoelectric focusing (IEF) in combination with mass spectrometry is described. Different types of substituted phenols of known pI values within the mass range 250-400 were used here as pI markers. The pure, synthesized pI markers were studied by MALDI-TOF/TOF MS. Fragmentation studies of the pI markers were also performed. Only stable and well-characterized pI markers were used in this work. The selected pI markers were mixed with proteins, deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of the focusing process and also estimation of the position of the invisible focused bands. The separated bands of the pI markers (containing separated proteins) were excised, and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. From the washed gel pieces the remaining carrier ampholytes were then washed out and proteins were in-gel digested with trypsin. The obtained peptides were measured by MALDI-TOF/TOF MS and the proteins identified via a protein database search. This procedure allows avoiding time-consuming protein staining and destaining procedures, which shortens the analysis time roughly by half. For comparison, IEF gels were stained with Coomassie Brilliant Blue R 250 and proteins in the gel bands were identified according to the standard proteomic protocol. This work has confirmed that our approach can give information about the correct pI values of particular proteins and shorten significantly the time of analysis.

A multivariate approach for the determination of isoelectric point of human carbonic anhydrase isoforms by capillary isoelectric focusing

“…Simultaneously, proteins focusing occurs in the pH zone corresponding to their p I values. Addition of natural 3, 8 or synthetic 9–12 p I markers within the CIEF gradient allows the precise determination of analyte p I . Several reviews on CIEF have been published, covering a wide spectrum of applications (p I determinations or interactions studies) on various samples (hemoglobin, monoclonal antibodies, microorganisms, viruses, tissues or proteomic biological matrix) 3–8.…”

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

“…However, MALDI-TOF/TOF MS can be also successfully applied to determination of other compounds including low-molecular-mass ones [20]. A limited set of low-molecular-mass pI markers selected on the basis of the mass spectrometric measurements of the pure compounds [21] was used in this work for simplification of measurements in the first implementation of this new method for proteomic identification of unknown proteins. In future, only colorless pI markers could be used because we plan to synthesize more pI markers and to cut the whole gel into equidistant pieces and analyze all of them.…”

Low‐molecular‐weight color pI markers to monitor on‐line the peptide focusing process in OFFGEL fractionation

“…Moreover, the low m/z range of precursor ions from m/z 250 to m/z 700 limited any interference from peptides in the MS analysis since the MS spectra of peptides were recorded within the mass range of 700–3500 m/z . Other different LMW color pI markers were previously used for the successful determination of protein pI values in gel electrofocusing (not including OGE) and mass spectrometry analysis, especially in complex mixtures. But, their mass spectra were more complex in terms of number of peaks and required a dual mass spectrometry analysis in positive and negative ion modes.…”