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首页> 外文期刊>Journal of mass spectrometry: JMS >Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides
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Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

机译：糖蛋白微异质性和宏观异质性的定量作图：使用合成肽和糖肽的质谱信号强度评估

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摘要

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICRMS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics.
机译：质谱（MS）用于量化连接到特定蛋白质糖基化位点的聚糖的相对分布（微异质性），并评估糖蛋白组学中的摩尔位点占有率（宏异质性）。但是，MS用于此类定量测量的准确性仍有待澄清。作为实现该目标的关键步骤，通过固相肽合成法化学合成了一组相关的胰蛋白酶肽，它们带有或不带有复杂的双天线二烯丙基化N-聚糖。合成模拟了使用PNGase F / A和内切D / F / H进行酶促去糖基化的肽，它们在糖基化位点分别带有天冬氨酸和N-乙酰氨基葡糖连接的天冬酰胺残基。使用各种质谱电离技术和质量分析仪（ESI-IT，ESI-Q-TOF，MALDI-TOF，ESI / MALDI-FT-ICRMS）研究了这些纯净，定义明确和定量的化合物的质谱电离/检测强度。取决于离子源/质谱分析仪，当分析等摩尔量时，带有复合型N-聚糖的糖肽显示出明显较低的信号强度（占非糖基化肽的10-50％）。当通过纳米ESI和中压MALDI分析糖肽时，观察到较小的电离/检测偏倚。胰蛋白酶肽中糖基化位点的位置也影响信号响应，特别是如果检测为单电荷或双电荷信号。这是第一项系统和定量解决和确定MS糖肽离子化/检测强度，以通过无标记方法评估糖蛋白微异质性和宏观异质性的研究。这些数据形成了准确定量糖蛋白组学急需的知识库。

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