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首页> 外文期刊>Journal of mass spectrometry: JMS >A convenient purification and preconcentration of peptides with alpha-cyano-4-hydroxycinnamic acid matrix crystals in a pipette tip for matrix-assisted laser desorption/ionization mass spectrometry
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A convenient purification and preconcentration of peptides with alpha-cyano-4-hydroxycinnamic acid matrix crystals in a pipette tip for matrix-assisted laser desorption/ionization mass spectrometry

机译：在移液器吸头中使用α-氰基-4-羟基肉桂酸基质晶体方便地纯化和预浓缩肽，用于基质辅助激光解吸/电离质谱

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摘要

Peptide samples derived from enzymatic in-gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed-phase pipette tip purification, on-target washing, adding co-matrices, etc. As a suitable matrix for MALDI MS of peptides, alpha-cyano-4-hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on-target washing of peptide samples can significantly improve MALDI MS signals. Although the common on-target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on-target washing principle carried out in a narrow-end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA-tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed.
机译：通过凝胶电泳解析的蛋白质通过酶促凝胶内消化获得的肽样品通常含有大量源自反应和分离缓冲液的盐。在基质辅助激光解吸/电离（MALDI）质谱（MS）之前，可以使用不同的方法进行脱盐，例如反相移液器吸头纯化，目标清洗，添加共基质等。作为肽MALDI MS的合适基质，经常使用α-氰基-4-羟基肉桂酸（CHCA）。 CHCA结晶显示了在其表面上结合肽的能力，并且由于它几乎不溶于酸性水溶液，因此，对肽样品的目标清洗可以显着改善MALDI MS信号。尽管常规的按目标清洗代表一种简单，便宜且快速的程序，但只有一小部分可用的肽溶液可有效地用于后续的MS分析。本方法结合了在窄端移液器吸头（例如GELoader吸头）中进行的按目标洗涤原理和通过使酸化溶液通过捕获在玻璃微纤维熔块上吸头内部的小型CHCA晶体对肽进行预浓缩的方法。使用CHCA-tip肽预浓缩进行MALDI MS分析的结果与使用自制POROS R2移液器吸头微柱可比。讨论了这种方法的优点和局限性。

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