Background

Sulfated glycans carrying sulfo sialyl LeX glycotope are ligands of L-selectin involved in lymphocyte homing in physiological and inflammatory states (Rosen, 2004; Kawashima, 2006). Many glycan array studies also showed that sulfated glycotopes are better ligands of several galectins (Ideo et al., 2002) and Siglecs (Bochner et al., 2005). However, there is very limited knowledge of endogenous sulfated glycans expressed on different types of mammalian cells, tissues or organism due to their low abundance and availability of few well-defined antibodies. Advanced mass spectrometry (MS)-based analysis is the only viable technique that would afford the requisite high sensitivity and accuracy for a meaningful probing of the cellular sulfoglycome.

Sulfated glycans are not readily detected in the conventional MS-based glycomic profiling. For analysis of non-derivatized, native sulfated glycans in the negative ion mode, the main problem is that sialylated glycans also carry negative charges and these are usually more abundant. Removing the sialic acids by treatment with sialidase is commonly applied (Hernandez Mir et al., 2009), but information on the sulfated sialylated glycans, which maybe the true physiological ligands for endogenous lectins is lost. For permethylated glycans, the advantages are neutralization of the negative charge of sialic acid and more reliable, sequence informative fragment ions can be obtained by MS/MS analyses. A precondition though is that the widely used sample clean-up method following permethylation, such as extraction by chloroform/water partition, which would result in loss of permethylated sulfated glycans into the aqueous layer, should be avoided (Morelle and Michalski, 2007; Kumagai et al., 2013).

We have shown that the conventional NaOH/DMSO slurry permethylation can retain the sulfates, and reverse phase (C18) solid phase extraction cartridge can be used to replace chloroform extraction (Yu et al., 2009; Khoo and Yu, 2010). Both permethylated non-sulfated and sulfated glycans are retained, extensively washed, and then eluted without introducing salts. MALDI-MS screening of permethylated glycans in the negative ion mode gives a quick survey for the presence of sulfated glycans. Instead of using 2,5 dihydroxybenzoic acid (DHB), which is a common matrix for glycan analysis in the positive ion mode (Harvey, 1993), we found that 3,4-diaminobenzophenone (DABP) afforded a better sensitivity for detecting permethylated sulfated glycans in the negative ion mode. Non-sulfated, mono-sulfated, di-sulfated permethylated glycans can be further fractionated by using a self-packed amine tip. Alternatively, a mixed mode weak anion exchange (MAX) cartridge containing both reverse phase and weak anion exchange properties can be used to replace the two-step process of cleaning and fractionation (Cheng et al., 2013; Cheng et al., 2015; Hsiao et al., 2017). The developed workflow for sulfoglycomics (Figure 1) has been successfully applied to many different biological samples such as lymph nodes from different glycosyltransferase/sulfotransferase knockout mice (Mitoma et al., 2007; Patnode et al., 2013), ovarian cancer (Shibata et al., 2012; Yu et al., 2013), colon cancer (Yu et al., 2018), immune cells (Patnode et al., 2013; Wang et al., 2013). Collectively, it showed that sulfated N- and O-glycans have a wider and ubiquitous occurrence than previously appreciated.


Figure 1. Schematic workflow for MS-based sulfoglycomic analyses. The key steps are reverse phase C18 which retains all permethylated glycans, and further fractionation of sulfated permethylated glycans from non-sulfated ones by amine beads. Alternatively, a mixed mode anion exchange (MAX) SPE is equally effective. MALDI-MS screening in the negative ion mode affords a quick profile of the sulfoglycome. This protocol is applicable to all commonly found sulfated N-glycans and O-glycans, irrespective of their reducing end status. The procedure for additional nanoLC-nanoESI-MS/MS analyses was described in another paper entitled “Negative Ion Mode nanoLC-ESI-MS/MS Analyses of Permethylated Sulfated Glycans” in Bio-protocol (Yu et al., 2020). SPE, solid phase extraction.