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

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.     

Surfactant self-assembling in gas phase: electrospray ionization- and matrix-assisted laser desorption/ionization-mass spectrometry of singly charged AOT clusters

The self-assembling of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in gas phase has been investigated by electrospray ionization- and matrix-assisted laser desorption/ionization mass spectrometry. Large surfactant clusters with an aggregation number close to that found in apolar media have been observed either as positive or negative ions. Moreover, the marked predominance of singly charged species as well as preliminary theoretical calculations strongly suggest an aggregate structure characterized by an internal hydrophilic core hosting the extra charge surrounded by an apolar shell constituted by the surfactant alkyl chains. This structure is similar to that of the more familiar reversed micelles formed when an appropriate surfactant is solubilized in apolar solvents. Finally, similar trends are observed independently either on the ionization technique or the polarity of the solvent used. This, together with the large dependence of the aggregation number on the flow rates, strongly indicates that self-assembling of the surfactant molecules occurs during the evaporation step.     

Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.     

Iron oxide/tantalum oxide core–shell magnetic nanoparticle-based microwave-assisted extraction for phosphopeptide enrichment from complex samples for MALDI MS analysis

A new type of metal-oxide-coated magnetic nanoparticles (NPs)—tantalum-oxide-coated magnetic iron oxide (Fe₃O₄@Ta₂O₅) NPs—which are used as affinity probes for selectively trapping phosphopeptides from complex samples, is demonstrated in this study. In this approach, phosphopeptide enrichment was achieved by incubating the NPs with sample solutions under microwave heating within 1 min. The NP–target species conjugates were readily isolated from samples by magnetic separation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. When using human serum as the sample, phosphorylated fibrinopeptide-A-derived ions are the only ions observed in the MALDI mass spectra after enrichment by the Fe₃O₄@Ta₂O₅ NPs. Furthermore, only phosphopeptides appear in the MALDI mass spectra after using the affinity probes to selectively trap target species from the tryptic digest of a cell lysate and milk sample. The results demonstrated that the Fe₃O₄@Ta₂O₅ NPs have the capability of selectively trapping phosphorylated peptides from complex samples. The detection limit of this approach for a phosphopeptide (FQpSEEQQQTEDELQDK) was ~10 fmol. Figure For the first time, tantalum oxide-coated magnetic iron oxide (Fe₃O₄@Ta₂O₅) NPs were demonstrated as suitable affinity-probes for selectively trapping phosphopeptides from complex samples. To shorten the analysis time, phosphopeptide enrichment was achieved by incubating the NPs with sample solutions under microwave-heating within 1 min. MALDI MS was employed for characterization of the species trapped by the NPs.