MS for glycopreteomics
质谱采集方法
目前尚无单一的质谱（MS）采集方法适用于所有类型的糖肽。根据所使用的质谱仪器类型，可选用多种采集策略。
Riley 等人对常用的质谱采集方法在多种 N-型和 O-型糖肽上的表现进行了基准评估[2020. Bertozzi._10.1021acs.jproteome.0c00218]。研究指出，根据糖基化类型的不同，以及是否需要实现糖基化位点定位或完整糖链结构解析，最适合的数据采集方式也会有所不同。
类似地，也有研究指出，对于新型或非典型糖肽的全面解析，仅采用单一的碎裂方式可能信息不全，而结合多种碎裂方法虽然会降低蛋白组覆盖度，但能够提供更全面的信息[Mol Omics, 16 (2020), 156]。
越来越多的研究开始尝试利用保留时间（retention time）预测等额外信息，来辅助糖肽的识别[J Proteome Res, 19 (2020), 2113]。这些方法通过整合不同糖链对液相洗脱行为的影响，提高了糖肽赋值的准确性。随着生物信息学工具的发展，预计未来保留时间信息将与 MS/MS 数据一起被更广泛地用于糖肽的识别与分析。
离子淌度质谱（ion-mobility MS）有助于分离异构结构（isomeric structures），从而获得更清晰的糖肽质谱图 [58,123,124]。
New instrumentation and novel fragmentation approaches
2019. Albert J.R. Heck. AC.
Simply Extending the Mass Range in Electron Transfer Higher Energy Collisional_10.1021acs.analchem.9b02125.pdf
Recently a simple yet extremely effective way to improve the identification of large glycopeptides was demonstrated by extending the mass range setting when analyzing EThcD scans within Orbitrap mass analyzers.
Extending the mass range beyond the standard settings enables the detection of highly informative high mass fragment ions, and was shown to improve the assignment confidence of N-linked glycopeptides by as much as 400-fold.
Although initially demonstrated for only N-linked glycopeptides, this simple alteration in data acquisition has also been used for O-linked glycopeptides[1],[2].
[1] 2020. Bertozzi. JPR. _10.1021acs.jproteome.0c00218.pdf
[2] 2020. Nichollas E. Scott. MCP. _10.1074mcp.TIR120.002100
2020. David J. Vocadlo, Jennifer S. Brodbelt. JACS
Precision Mapping of O-Linked N-Acetylglucosamine Sites in Proteins Using_10.1021jacs.0c04710.pdf
Alternative fragmentation approaches are also being explored to improve the identification of glycopeptides. An example of this is the recent demonstration of the ability of ultraviolet photodissociation (UVPD) fragmentation to enable precise localization of O-GlcNAc events [55].
2019. Joshua J. Coon. Nat Commun.
Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis_10.1038s41467-019-09222-w.pdf
activated ion electron transfer dissociation (AI-ETD), a hybrid fragmentation method where ions are concurrently activated by infrared irradiation in addition to undergoing ETD, was shown to enable deep glycoproteomic analysis outperforming previously reported acquisition approaches.
This approach has now been utilized to compare glycoproteomes of isogenic cell lines, enabling the identification of 1000s of N-glycopeptides and the quantification of surface proteome changes in response to oncogenic transformation[2020. James A. Wells. PNAS. _10.1073pnas.1917947117].
New data-acquisition and glycopeptide detection methods
IsoTaG
the isotope targeted glycoproteomics (IsoTaG) approach uses MS1 based information to identify glycopeptides. The IsoTaG approach exploits the addition of isotope tags containing 79Br and 81Br atoms into metabolically labelled glycopeptides, resulting in glycopeptides having a unique mass envelope compared to non-labelled peptides.
Recently IsoTaG was shown to enable the identification of over 2000 O-GlcNAc glycopeptides from ∼1 mg of material [2018. Christina M. Woo. MCP. _10.1074mcp.RA117.000261].
However, IsoTaG requires multiple LC–MS runs and specialized software (IsoStamp[ACS Chem Biol 2011, 6:829]) to generate the inclusion lists needed to select potential IsoTaG glycopeptides for identification.
DIA
数据非依赖采集（data-independent acquisition, DIA）在N-连接型（N-linked）[Mol Cell Proteomics 2020, 19:672；J Proteomics 2018, 172:68]和O-连接型（O-linked）[Nat Methods 2019, 16:902]糖肽样本研究中的应用日益广泛。
需要注意的是，尽管可以通过DIA监测O-连接型糖肽，但这通常是借助高能碰撞诱导解离（HCD）碎裂实现的，而HCD并不总能实现糖基化位点的明确鉴定。尽管目前一代的质谱仪器已支持以DIA方式进行电子转移解离（ETD）或ETD加高能碰撞（EThcD）碎裂，但由于这些碎裂方法的速度较慢，使得在DIA中应用ETD/EThcD在技术上仍存在挑战。
Glycopeptide enrichment independent analysis
无需糖肽富集的分析方法
尽管糖肽富集策略在糖蛋白质组研究中被广泛应用，但随着仪器分析速度和灵敏度的提升，越来越多的研究开始采用不依赖富集的分析方法。在这些方法中，可以在一次分析中同时获得蛋白质组和糖蛋白质组数据，从而能够监测如糖基化位点占据率（site occupancy）等性质。这种分析方式可以在蛋白丰度的背景下理解糖基化变化。
这类研究的示例之一是近期的一项工作，巧妙地利用脉冲稳定同位素标记（pulse stable isotope labelling）来追踪模型蛋白IgG上的位点特异性N-糖基化（site-specific N-glycosylation）加工过程 [Sci Adv 2019, 5:eaax8930]。这种分析方法使得研究人员能够以时间为变量监测IgG上的N-聚糖（N-glycan）加工过程，并据此建立了蛋白质N-聚糖加工的数学模型。
类似地，不依赖糖肽富集的方法也被用于研究酵母糖蛋白质组（yeast glycoproteomes）[Methods Mol Biol 2019, 2049:191]，以探索富含糖蛋白的细胞壁中糖基化占据率的变化。
迄今为止，这类不依赖糖肽富集的方法通常应用于单一蛋白或复杂度有限的蛋白质组。然而，随着分析仪器的进一步发展，这种分析方式可能会被研究社区更广泛地采用。
