MALDI‑2 for the Enhanced Analysis of N‑Linked Glycans by Mass Spectrometry Imaging

N-glycans are important players in a variety of pathologies including different types of cancer, (auto)­immune diseases, and also viral infections. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important tool for high-throughput N-glycan profiling and, upon use of tandem MS, for structure determination. By use of MALDI-MS imaging (MSI) in combination with PNGase F treatment, also spatially correlated N-glycan profiling from tissue sections becomes possible. Here we coupled laser-induced postionization, or MALDI-2, to a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF fleX MALDI-2, Bruker Daltonics). We demonstrate that with MALDI-2 the sensitivity for the detection of molecular [M – H]− species of N-glycans increased by about 3 orders of magnitude. Compared to the current gold standard, the positive ion mode analysis of [M + Na]+ adducts, a sensitivity increase by about a factor of 10 is achieved. By exploiting the advantageous fragmentation behavior of [M – H]− ions, exceedingly rich structural information on the composition of complex N-glycans was moreover obtained directly from thin tissue sections of human cerebellum and upon use of low-energy collision-induced dissociation tandem MS. In another set of experiments, in this case by use of a modified Synapt G2-S QTOF mass spectrometer (Waters), we investigated the influence of relevant input parameters, in particular pressure of the N2 cooling gas in the ion source, delay between the two laser pulses, and that of their pulse energies. In this way, analytical conditions were identified at which molecular ion abundances were maximized and fragmentation reactions minimized. The use of negative ion mode MALDI-2-MSI could constitute a valuable tool in glycobiology research.N-聚糖（N-glycans）是参与多种病理过程的关键分子，涵盖不同类型癌症、（自身）免疫疾病以及病毒感染。基质辅助激光解吸电离质谱（MALDI-MS）是实现高通量N-聚糖谱图分析的重要工具，结合串联质谱（tandem MS）可完成结构鉴定。若将MALDI质谱成像（MALDI-MS imaging, MSI）与PNGase F酶处理联用，还可实现组织切片的空间相关性N-聚糖谱图分析。本研究将激光诱导后电离技术（又称MALDI-2）与捕获离子迁移率四极杆飞行时间质谱仪（timsTOF fleX MALDI-2，布鲁克道尔顿（Bruker Daltonics））联用。研究表明，采用MALDI-2技术时，N-聚糖的分子[M – H]⁻离子检测灵敏度提升了约3个数量级；相较于当前的金标准——[M + Na]⁺加合物的正离子模式分析，本方法的灵敏度进一步提升约10倍。借助[M – H]⁻离子优异的碎裂行为，研究团队还可直接从人类小脑薄组织切片中获取极为丰富的复杂N-聚糖组成结构信息，且仅需采用低能碰撞诱导解离串联质谱即可实现。在另一组实验中，本研究采用改装后的Synapt G2-S QTOF质谱仪（沃特世（Waters）），探究了相关输入参数的影响，具体包括离子源内N₂冷却气体压力、两次激光脉冲间的延迟时间以及脉冲能量。借此确定了可使分子离子丰度最大化、碎裂反应最小化的分析条件。负离子模式MALDI-2-MSI的应用有望成为糖生物学研究中的有力工具。