Abstract:

A method for the fast separation of human serum and enrichment of low abundance proteins was developed using offline 2D liquid chromatography (2D-LC) consisted of chromatographic cake (10 mm×20 mm i.d.) and reversed-phase liquid chromatography (RPLC). The protein after separation and enrichment was detected using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). This method was validated by four standard proteins at very low concentration. It was found that the detection limits were 1 pmol/μL for the enriched cytochrome-c and myoglobin, and 0.1 pmol/μL for enriched lysozyme and insulin. This method has been applied to the proteomic research of human serum, and it was found that the signal intensity and the number of detected proteins/peptides in MALDI-TOF MS increased with the increase of the loading sample volume of the human serum on chromatographic cake. A total of 285 fractions (Mr<15000) were found when 1.0 mL serum sample was loaded on the chromatographic cake. In addition, cytochrome-c in low abundance was also separated and enriched successfully when 1 μg cytochrome-c was added into 0.5 mL original serum. The results showed that 2D-LC consisting of the chromatographic cake and RPLC was successfully applied to not only the fast separation and preparation of human serum sample with large loading volume in one cycle of analysis, but also the efficient isolation and enrichment of the lower abundance proteins/peptides in human serum. Moreover, it successfully increased the detection efficiency of the low abundance proteins/peptides in human serum with MALDI-TOF MS.

Key words: chromatographic cake, human serum , matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), proteomics, 2D-liquid chromatography (2D-LC)