Improved two-dimensional electrophoretic mapping of Japanese human hair proteins; application to curved and straight Japanese human hairs; and protein identification by MALDI MS and MS/MS quadrupole time-of-flight mass spectrometry

Objectives: To evaluate improved protein extraction and two‐dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women’s human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. Methods: Hair keratin and keratin‐associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2‐carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric‐focused on first‐dimensional pH gradient gel strips, then separated by molecular weight on laboratory‐made, second‐dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty‐eight proteins from a JRH 2DE gel were enzyme‐cleaved for MALDI‐TOF‐MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in‐house human hair protein database incorporating ninety‐eight annotated protein sequences assisted MS analysis. Results: 2DE gels of tris(2‐carboxyethyl) phosphine‐extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol‐extracted JRH and published commercially made second‐dimensional gels. Silver‐stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over‐staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty‐nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG‐stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. Conclusion: Root‐end human hair extraction with tris(2‐carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight.