Quantitative Determination of the Series of Blood Serum Markers Without Its Preliminary Fractionation Using the Special Features of Tripsine Interaction With Alpha-2-Macroglobulin Using Maldi-MS

Abstract

Предложен способ модификации мишени для МАLDI-МS-анализа, позволяющий селективно выделять аналиты из биологических образцов непосредственно на поверхности мишени, как альтернатива классическим методам. Для модификации мишени суспензию металл-аффинного сорбента на основе оксида железа(III) в 50% водно-метанольном растворе электрораспыляли в бескапельном режиме c динамическим делением потока жидкости при атмосферном давлении в нормальных условиях. МАLDI-мишень выступала в качестве противоэлектрода. На МАLDI-мишень наносился слой сорбента в виде пятна, частицы которого в дальнейшем устойчивы к воздействию растворителей. На модифицированной МАLDI-мишени проводилось металл-аффинное обогащение фосфорилированного пептида с аминокислотной последовательностью SSNGHV(pY)EKLSSI из образца триптического гидролизата глобина человека. МАLDI-масс-спектр записывали с пятна сорбента. Такая методика создана в качестве альтернативы трудоемкой пробоподготовке биопроб и позволяет ограничиться минимальными объемами образца и растворителей.

Quench me if you can: Alpha-2-macroglobulin trypsin complexes enable serum biomarker analysis by MALDI mass spectrometry

“…Since the proteolytic activity of trypsin in complex with A2M is limited, and it loses the ability to hydrolyze major serum proteins (for example, albumin) due to their large sizes (well above 20 kDa), such A2M-trypsin complexes may be useful as a research tool for identifying minor, small serum proteins. It has been shown earlier that serum amyloid A (SAA) fragments can be detected in mass spectra after serum treatment with trypsin for two hours [8,20]. Thus, it was observed in our experiments that some other ions, that differ from SAA, appear in the mass spectrum with longer incubation of serum with trypsin (marked by asterisk in Fig.…”

“…In our previous study [20], the possibility of A2M concentration assessment, directly in human blood serum, was demonstrated. Briefly, it was shown that: after addition of trypsin to serum and a few minutes of incubation, an intense, singly-protonated quasimolecular ion ( m / z = 1259 .…”

Quench me if you can: Alpha-2-macroglobulin trypsin complexes enable serum biomarker analysis by MALDI mass spectrometry

“…690-728), as expected; this region is involved in trypsin binding and inactivation by A2M in blood serum. It was also shown that the amount of peptide detected is equal to the amount of trypsin bound with A2M [20].…”

“…In our previous study [20], the possibility of A2M concentration assessment, directly in human blood serum, was demonstrated. Briefly, it was shown that: after addition of trypsin to serum and a few minutes of incubation, an intense, singlyprotonated quasimolecular ion ( ⁄ = 1259.57) is registered in the MALDI mass spectrum.…”

“…Our approach is based on peculiarities of the A2M/trypsin interaction, which results in the appearance of a characteristic peptide, VGFYESDVMGR [1]. This peptide is easily detectable by MALDI-MS, and its concentrations is proportional to A2M in blood serum [20].…”