Proteomics often exploits sequential cutting of the SDS-PAGE gel lane into slices with subsequent identification of proteins by peptide mass fingerprinting (PMF). In this paper, the influence of slice thickness on protein identification was investigated. Following the separation of the human liver microsomal fraction, the 37 to 75 kDa range of the gel lane was cut into 20 or 40 slices of 0.5 mm and 0.25 mm thickness, respectively. Identification of proteins after trypsinolysis was performed by MALDI-TOF mass spectrometry. A twofold reduction of slice thickness did not influence the number of peaks in PMF-spectra. It was established that at a tolerance of 0.15 Da the number of identified proteins in the 40 slices series exceeded by more than twofold the number of identifications in 20 slices series. It was shown that the decrease of slice thickness leads to a considerable enhancement of peptide peak's intensity and, also, to the changes of PMF-spectra which resulted gaining additional peptides for enrichment of sequence coverage.

Using electrospray ionization tandem mass spectrometry, a comparative analysis of the HaCaT keratinocyte proteins encoded by the 18th chromosome was performed before and after exposure to sodium dodecyl sulfate (25 mg/ml) and to Triton X-100 (12.5 mg/ml) in a subtoxic dose for 48 hours. Proteins were identified using the SearchGUI platform (X!Tandem and MS-GF+ search engines). In total, 1284 proteins were found in immortalized human HaCaT keratinocytes and about 75% of them were identified by two or more peptides. Were identified, that 26 proteins were encoded by genes of chromosome 18. Among these proteins, 17 were common for control cells and HaCaT cells treated with SDS. Proteins MARE2 and CTIF were identified only in control keratinocytes. Seven identified proteins encoded by genes of chromosome 18 were found only in detergent-treated keratinocytes: LMAN1, NDUV2, SPB3, VPS4B, KDSR, ROCK1 and RHG28.

BackgroundVitellogenin (Vtg) is the major egg yolk protein (YP) in most oviparous species and may be useful as an indicator in ecotoxicological testing at the biochemical level. In this study, we obtained detailed information about the Vtgs of Danio rerio embryos by cutting SDS-PAGE gel lanes into thin slices, and analyzing them slice-by-slice with (MALDI-TOF) mass spectrometry.ResultsWe conducted three proteomic analyses, comparing embryonic Danio rerio Vtg cleavage products after exposure for 48 h to CdSecore/ZnSshell quantum dots (QDs), after exposure to a mixture of the components used for quantum dot synthesis (MCS-QDs), and in untreated embryos. The Vtg mass spectrometric profiles of the QDs-treated embryos differed from those of the unexposed or MCS-QDs-treated embryos.ConclusionThis study demonstrates the possible utility of Vtg profiling in D. rerio embryos as a sensitive diagnostic tool to estimate nanoparticle toxicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0072-7) contains supplementary material, which is available to authorized users.

The effects of phosphatidylcholine-based phospholipid nanoparticles containing fullerene C60 on Danio rerio fish embryos were studied. Exposure of the embryos with the nanoparticles for 48 h did not lead to appreciable changes in the number of protein bands in SDS-PAGE in comparison with the control (exposure in medium with phosphatidylcholine). Mass spectrometric identification of proteins showed differences in the proteomic profiles of the samples. The content of vitellogenins changed after exposure with phosphatidylcholine-based nanoparticles with C60 fullerenes. This could indicate low toxicity of the nanoparticles towards D. rerio embryos under experimental conditions.

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