Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry
Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry
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DOI：
10.1021/ac0503963
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发表时间：
2005-07-15
影响因子：
7.4
通讯作者：
Zubarev, RA
中科院分区：
文献类型：
--
作者：
Adams, CM;Zubarev, RA
利用电子捕获离解（ECD）和碰撞激活离解（CAD）的串联质谱（MS/MS）技术，建立了一种定性和定量分析多肽中单个氨基酸残基的手性分析方法。ECD比CAD产生了更明显的手性识别，这是由于ECD的振动激发程度较小。在这项研究中使用了几种肽和蛋白质模型系统，包括已知最小的蛋白质，色氨酸笼，乳铁蛋白肽，以及生物学上相关的阿片肽，皮啡肽。采用了一种改进的动力学方法来量化立体异构体多肽片段模式之间的分离程度，并将其作为片段离子丰度的函数。所获得的非对映异构体肽混合物中d -氨基酸相对丰度的校准尺度在ECD中精确到1%，在CAD中精确到3-5%。研究发现，纳米流反相液相色谱法可以很好地进行立体异构体的分离和定量，而在线MS/MS的目的仅限于立体异构体的鉴定。该技术有望用于多肽和蛋白质的手性取代分析，拓宽了串联质谱分析的应用领域。
Tandem mass spectrometry (MS/MS) utilWng both electron capture dissociation (ECD) and collisionally activated dissociation (CAD) was used to develop a qualitative and quantitative analytical method for chiral analysis of individual amino acid residues in polypeptides. ECD produced a more distinct chiral recognition than CAD, which is attributed to the smaller degree of vibrational excitation in ECD. Several peptide and protein model systems were used in this study, including the smallest known protein, tryptophan cage, a Iactoferrin peptide, and the biologically relevant opioid peptide, dermorphin. An adaptation of the kinetic method was used to quantify the degree of separation between fragmentation patterns of stereoisomeric peptides as a function of fragment ion abundances. The obtained calibration scale for relative abundances of D-amino acids in diastereomeric peptide mixtures was accurate to 1% for ECD and to 3-5% for CAD. It was found that separation and quantification of stereoisomers could be advantageously performed by nanoflow reversed-phase liquid chromatography, with the objective of online MS/MS limited to stereoisomer identification. This technique shows promise for the analysis of chiral substitution in peptides and proteins, broadening the application area for tandem mass spectrometry.