Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery
Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery
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DOI：
10.1021/pr050046x
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发表时间：
2005-05-01
影响因子：
4.4
通讯作者：
Kobayashi, R
中科院分区：
文献类型：
--
作者：
Koomen, JM;Li, DH;Kobayashi, R
低分子量血浆蛋白质组及其生物学相关性尚未明确定义；因此，进行了直接测序和鉴定血浆和血清蛋白谱中观察到的肽的实验。使用蛋白质分级分离、基质辅助激光解吸电离质谱 (MALDI-MS) 分析以及液相色谱法与 MALDI 串联质谱 (MS/MS) 测序相结合来分析肝素化血浆的低分子量蛋白质组。使用功能衍生化 96 孔板的四种分级分离技术用于从血浆中提取肽。串联 TOF 在事先不了解序列的情况下成功识别了 m/z 5500 的肽，并且还用于验证较大离子信号的序列分配。这些谱中测序的肽 (n > 250) 来自数量惊人的少量蛋白质 (n 大约 20)，这些蛋白质都是血浆常见的，包括纤维蛋白原、补体成分、抗蛋白酶和载体蛋白。裂解模式与已知血浆蛋白酶的裂解模式一致，包括凝血酶、纤溶酶和补体蛋白的初始裂解，随后是氨肽酶和羧肽酶活性。在这些数据的基础上，我们讨论了使用粗分级分离和 MALDI-MS 分析在低分子量血浆或血清蛋白质组中发现生物标志物的局限性。
The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/ MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n > 250) sequenced in these profiles came from a surprisingly small number of proteins (n approximate to 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.