NSW Systems Biology Initiative, University of New South Wales, Sydney, New South Wales 2052, Australia.
J Am Soc Mass Spectrom. 2012 Jan;23(1):124-40. doi: 10.1007/s13361-011-0273-y. Epub 2011 Nov 15.
Broad-scale mass spectrometric analyses of glycopeptides are constrained by the considerable complexity inherent to glycoproteomics, and techniques are still being actively developed to address the associated analytical difficulties. Here we apply Orbitrap mass analysis and higher-energy C-trap dissociation (HCD) to facilitate detailed insights into the compositions and heterogeneity of complex mixtures of low abundance glycopeptides. By generating diagnostic oxonium product ions at mass measurement errors of <5 ppm, highly selective glycopeptide precursor ion detections are made at sub-fmol limits of detection: analyses of proteolytic digests of a hen egg glycoprotein mixture detect 88 previously uncharacterized glycopeptides from 666 precursor ions selected for MS/MS, with only one false positive due to co-fragmentation of a non-glycosylated peptide with a glycopeptide. We also demonstrate that by (1) identifying multiple series of glycoforms using high mass accuracy single stage MS spectra, and (2) performing product ion scans at optimized HCD collision energies, the identification of peptide + N-acetylhexosamine (HexNAc) ions (Y1 ions) can be readily achieved at <5 ppm mass measurement errors. These data allow base peptide sequences and glycan compositional information to be attained with high confidence, even for glycopeptides that produce weak precursor ion signals and/or low quality MS/MS spectra. The glycopeptides characterized from low fmol abundances using these methods allow two previously unreported glycosylation sites on the Gallus gallus protein ovoglycoprotein (amino acids 82 and 90) to be confirmed; considerable glycan heterogeneities at amino acid 90 of ovoglycoprotein, and amino acids 34 and 77 of Gallus gallus ovomucoid are also revealed.
广泛的糖肽质谱分析受到糖蛋白质组学固有复杂性的限制，目前仍在积极开发技术来解决相关的分析难题。在这里，我们应用 Orbitrap 质谱分析和更高能量 C 阱解离（HCD）技术，深入了解低丰度糖肽复杂混合物的组成和异质性。通过在质量测量误差<5 ppm 时生成诊断性氧鎓产物离子，可在亚 fmol 检测限下实现高度选择性的糖肽前体离子检测：对鸡卵蛋白混合物的酶解产物进行分析，从 666 个用于 MS/MS 的前体离子中检测到 88 个以前未表征的糖肽，仅由于非糖基化肽与糖肽的共碎裂而产生一个假阳性。我们还证明，通过（1）使用高质量准确度单级 MS 谱识别多个系列的糖型，以及（2）在优化的 HCD 碰撞能量下进行产物离子扫描，可以在<5 ppm 的质量测量误差下轻松识别肽+N-乙酰己糖胺（HexNAc）离子（Y1 离子）。这些数据允许以高置信度获得基肽序列和聚糖组成信息，即使对于产生弱前体离子信号和/或低质量 MS/MS 谱的糖肽也是如此。使用这些方法从低 fmol 丰度表征的糖肽允许确认 Gallus gallus 蛋白卵粘蛋白（氨基酸 82 和 90）上两个以前未报道的糖基化位点；还揭示了卵粘蛋白中氨基酸 90 和 Gallus gallus 卵转铁蛋白中氨基酸 34 和 77 的大量聚糖异质性。
