Advancing a high throughput glycotope-centric glycomics workflow based on nanoLC-MS2-product dependent-MS3 analysis of permethylated glycans
Rationale: The intrinsic nature of glycosylation, namely non-template encoded, stepwise elongation and termination with a diverse range of isomeric glyco-epitopes (glycotopes), translates into ambiguity in most cases of mass spectrometry (MS)-based glycomic mapping. Rapid mapping by MS analysis often provides only a coarse sketch of the glycomic complexity based primarily on glycosyl compositions, whereby the missing high-resolution structural details require a combination of multi-mode separations and multi-stages of induced fragmentation to gain sufficiently discriminative precision, often at the expenses of throughput and sensitivity. It is arguable that whether one needs to delineate every single glycomic entity, which may be counterproductive. Instead, one should focus on identifying as many structural features as possible that would collectively define the glycomic characteristics of a cell or tissue, and how these may change in response to self-programmed development, immuno-activation, and malignant transformation.
Given the available technology and foreseeable advances in the near future, homing in on resolving the terminal fucosylated, sialylated and/or sulfated structural units, or glycotopes, maybe a more pragmatic and ultimately more rewarding approach to gain insights into myriad biological processes mediated by these terminal coding units carried on important glycoproteins, to be decoded by a host of endogenous glycan-binding proteins and antibodies. To prioritize analytical focus on the more tangible glycotopes is akin to first identifying the eye-catching and characteristic-defining flowers and fruits of the glyco-forest, to see the forest for the trees. It has the best prospects of attaining the much-needed balance in sensitivity, structural precision and analytical throughput to match advances in other omics. [A mass spectrometry-based glycotope-centric cellular glycomics is the more fruitful way forward to see the forest for the trees. (2021). Khoo KH. Biochem Soc Trans 49(1): 55-69.]
Given the available technology and foreseeable advances in the near future, homing in on resolving the terminal fucosylated, sialylated and/or sulfated structural units, or glycotopes, maybe a more pragmatic and ultimately more rewarding approach to gain insights into myriad biological processes mediated by these terminal coding units carried on important glycoproteins, to be decoded by a host of endogenous glycan-binding proteins and antibodies. To prioritize analytical focus on the more tangible glycotopes is akin to first identifying the eye-catching and characteristic-defining flowers and fruits of the glyco-forest, to see the forest for the trees. It has the best prospects of attaining the much-needed balance in sensitivity, structural precision and analytical throughput to match advances in other omics. [A mass spectrometry-based glycotope-centric cellular glycomics is the more fruitful way forward to see the forest for the trees. (2021). Khoo KH. Biochem Soc Trans 49(1): 55-69.]
- We have been pursuing analytical strategy that homes in on identifying the terminal sulfo-, sialyl and/or fucosylated glycotopes by comprehensive nanoLC-MS2-product dependent MS3 analysis of permethylated glycans, in conjunction with development of a data mining computational tool, GlyPick, to enable an automated, high throughput, semi-quantitative glycotope-centric glycomic mapping amenable to even non-experts [Hsiao et al, Mol Cell Proteomics 16(12): 2268-2280].
- We demonstrated that diagnostic MS2 ions can be relied upon to inform the presence of specific glycotopes, whereas their possible isomeric identities can be resolved at MS3 level. This includes the sulfated glycotopes to be delineated in negative ion mode for sulfoglycomics [Cheng et al., Anal Chem 87(12):6380-6388]
- The high acquisition speed, resolution, and mass accuracy afforded by top-notch Orbitrap Fusion MS system now allow a sensible spectral count and/or summed ion intensity-based glycome-wide glycotope quantification.
- An added advantage is that such an analytical approach utilizes the same acidic reverse phase C18 nanoLC conditions fully compatible with proteomic analysis to allow rapid hassle-free switching.
