Trimpin Sarah, Deinzer Max L
Oregon Health and Science University, CROET, Portland, Oregon 97239, USA.
J Am Soc Mass Spectrom. 2007 Aug;18(8):1533-43. doi: 10.1016/j.jasms.2007.04.017. Epub 2007 Apr 29.
Manual and automated solvent-free mini-ball mill (MBM) matrix-assisted laser desorption/ionization (MALDI) analysis of mixtures of beta-amyloid peptides (1-11), (33-42), (1-42) and non-beta-amyloid component of Alzheimer's disease peptide yielded interpretable spectra for all of the peptides present regardless of their relative amounts in the samples. This was not the case for solvent-based MALDI analysis using traditional acidic aqueous/organic solvent conditions, which resulted in severe over-representation of hydrophilic peptide (1-11) and provided no spectra for insoluble amphiphilic peptide (1-42) even when present at 50% relative molar amount. Less accurate representation of components in mixtures by the traditional method appears to be a combination of poor dissolution of peptides in the solvent and preferential ionization of more hydrophilic peptides in the mixture. Consequently, only MBM provided a complete tryptic map of beta-amyloid (1-42) compared to 67% coverage by traditional MALDI. Acetonitrile (0.1% TFA) led to improved coverage only at a 50% molar ratio of peptide (1-42), but also to a side product of (1-42), Met oxidation (amino acid 35), a phenomenon not observed in MBM MALDI analysis. Traditional MALDI analysis resulted in over-representation of hydrophilic soluble beta-amyloid (1-11) in defined mixtures and autoproteolytic peptides of trypsin. In contrast, over-representation and under-representation were less pronounced in solvent-free MALDI in all of the investigated cases. Analysis of defined peptide and tryptic peptide mixtures showed that MBM MALDI yielded greater qualitative reliability, which also improved quantitative response relative to the solvent-based approach.
对β-淀粉样肽（1-11）、（33-42）、（1-42）混合物以及阿尔茨海默病肽的非β-淀粉样成分进行手动和自动无溶剂微型球磨仪（MBM）基质辅助激光解吸/电离（MALDI）分析，无论样品中各肽的相对含量如何，均可得到所有存在肽的可解释光谱。而使用传统酸性水/有机溶剂条件的基于溶剂的MALDI分析则并非如此，该方法导致亲水性肽（1-11）严重过度呈现，即使两亲性不溶性肽（1-42）以50%的相对摩尔量存在时也无法得到其光谱。传统方法对混合物中各成分的呈现不够准确，这似乎是由于肽在溶剂中溶解不佳以及混合物中亲水性更强的肽优先电离所致。因此，与传统MALDI的67%覆盖率相比，只有MBM能提供β-淀粉样肽（1-42）完整的胰蛋白酶图谱。乙腈（0.1%三氟乙酸）仅在肽（1-42）摩尔比为50%时能提高覆盖率，但同时也会产生（1-42）的副产物，即甲硫氨酸氧化（氨基酸35），而在MBM MALDI分析中未观察到这种现象。传统MALDI分析导致在特定混合物中亲水性可溶性β-淀粉样肽（1-11）和胰蛋白酶的自蛋白水解肽过度呈现。相比之下，在所有研究案例中，无溶剂MALDI中过度呈现和呈现不足的情况都不那么明显。对特定肽和胰蛋白酶肽混合物的分析表明，MBM MALDI具有更高的定性可靠性，相对于基于溶剂的方法，其定量响应也有所改善。
