Merrell Karen, Thulin Craig D, Esplin M Sean, Graves Steven W
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA.
Rapid Commun Mass Spectrom. 2009 Sep;23(17):2685-96. doi: 10.1002/rcm.4168.
The low-abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low-abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time-of-flight MS instrument. As a demonstration of the approach, two low-abundance peptides with masses of approximately 4000-5000 Da were selected for MS/MS sequencing. The multi-channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279 Da and 5061 Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C-terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery.
低丰度、低分子量血清蛋白质组在利用质谱（MS）发现新生物标志物方面具有很高潜力。由于血清蛋白质组庞大且复杂，确定比较组之间某一分子物种的相对定量差异需要一种具有强大分离能力、高灵敏度以及高质量分辨率的方法。毛细管液相色谱（cLC）/MS既提供了必要的分离技术，又具备观察许多低丰度肽段的灵敏度。原则上，在cLC/MS步骤中观察到的潜在血清肽生物标志物，后续可通过串联cLC/MS/MS进行鉴定，无需进一步样品制备或额外仪器。在本报告中，描述了一种用于肽测序的新型cLC/MS/MS方法，该方法超越了先前报道的使用串联飞行时间质谱仪通过碰撞碎裂完成氨基酸测序的大小限制。作为该方法的一个实例，选择了两个质量约为4000 - 5000 Da的低丰度肽段进行MS/MS测序。多通道分析仪（MCA）以一种新颖的方式使用，允许对几种定制碰撞能量下的每一个肽段求和120个碎裂谱图，从而以改善的信噪比提供每个肽段更全面的碎裂覆盖。将该复合分析得到的峰列表提交给Mascot进行鉴定。成功鉴定出两个索引肽段，分子量分别为4279 Da和5061 Da。这些肽段分别是一个39个氨基酸的免疫球蛋白G重链可变区片段和一个47个氨基酸的纤维蛋白α异构体C末端片段。本文所述方法既能够检测数千种血清分子，又能与显著增强的cLC/MS/MS肽测序能力相结合，为血清生物标志物发现提供了一种有前景的技术。
