**2.1 Peptide generation**

To generate peptides for proteomics analyses, a protein sample is first digested with a protease. The specificity of the protease used can significantly impact the protein coverage obtained by the method. Trypsin, a protease that cleaves after lysine and arginine residues, has been the workhorse of the proteomics field for over two decades. Trypsin generally produces peptides of sufficient length to ionize efficiently in mass spectrometry. However, protein-specific challenges can occur with trypsin, especially with glycoproteins. For example, some proteins naturally lack lysine or arginine residues, have these residues disparately positioned, or have bulky glycans in close proximity that sterically hinder proteolysis. Each of these factors can produce larger peptides that typically do not ionize as well. As such, other proteases with cleavage specificities orthogonal to trypsin are often used to increase proteolytic peptide coverage (**Table 1**) [17]. For example, **Figure 3** shows that α-Lytic Protease can be used alone or in combination with other proteases to yield increased sequence coverage.

A recent advance has been the use of O-glycan-specific proteases (O-endoproteases) for generating O-glycopeptides for analysis. These enzymes recognize and bind to mucin-type O-glycans, then cleave the peptide bond immediately N-terminal to the glycosylated serine or threonine. Used either alone or in series with other proteases like trypsin, glycopeptides are generated that have an O-glycan on their amino-terminal amino acid following cleavage. The first commercial enzyme of this class was the O-endoprotease from *Akkermansia muciniphila* (sold under the trade name OpeRATOR, Genovis AB, Sweden). This enzyme recognizes mammalian O-glycans but it is inhibited by the presence of terminal sialic acids. Accordingly, sialidase treatment is required for efficient performance which results in loss of glycan structural information. Recently, chemical modification of sialic acids has also been shown to improve OpeRATOR function [18]. In contrast, the O-glycoprotease newly available from New England Biolabs, is not inhibited by the presence of sialic acids and it also exhibits a broad specificity towards proteins with mammalian


#### **Table 1.**

*Proteases used in proteomics. Protease specificities are indicated using single letter codes for amino acid residues. Recognition sites that are cleaved at a lower rate are indicated by amino acids bracketed by parentheses.*

#### **Figure 3.**

α*-Lytic Protease can be used alone or in combination with other proteases to yield increased sequence coverage. Comparison of sequence coverage for three protein standards after parallel digestion using Trypsin (blue) or a-Lytic Protease (gold). The combined data set (grey) results in overlapping peptides and increased sequence coverage. (Reprinted by permission from New England Biolabs. https://www.neb.com/products/ p8113-a-lytic-protease#Product%20Information).*

O-glycans. This enzyme recognizes O-glycans ranging in size from a minimal GalNAc-α-Ser/Thr structure to larger mucin-type O-glycans bearing branches and sialic acids. This specificity negates the need for sialidase treatment or chemical modification prior to O-glycopeptide generation. Resulting O-glycopeptides can be mapped to identify the protein of origin, the position of O-glycosites, and the range of O-glycan structures present at any given glycosite in a single experiment.

### **2.2 Glycopeptide enrichment methods**

Glycopeptides are typically in low abundance compared to aglycosylated peptides in a peptide mixture. Additionally, it is well-established that ionization of glycopeptides is often weaker compared to aglycosylated peptides during MS analyses [19]. This results in aglycosylated peptide signals often dominating MS experiments. Therefore, enrichment of glycopeptides prior to sample analysis has been a growing trend to improve intact glycopeptide identification. Several enrichment schemes that vary in their rationales have been described. These approaches range from general methods (enrichment of both N- and O-linked glycopeptides) to newer glycan class-specific approaches that selectively enrich for either N- or O-linked glycopeptides. Several approaches are summarized here.

### *2.2.1 Hydrophilic interaction liquid chromatography (HILIC)*

HILIC has been widely used for glycopeptide enrichment. It is based on the interaction between the hydrophilic glycan moiety of a glycopeptide and the polar stationary phase in the non-polar mobile phase (typically acetonitrile). Many HILIC materials have been developed, however zwitterionic HILIC (ZIC-HILIC) enrichment is generally the most useful due to higher loading capacity and broader specificity. HILIC does not discriminate between O-linked and N-linked glycopeptides and hydrophilic non-glycosylated peptides may co-elute [20]. Thus, for more complete glycopeptide enrichment, HILIC may require a complementary chromatography fractionation step [21].

### *2.2.2 Boronic acid*

One method utilizes boronic acid presented on a solid support to react with cis-diol-containing saccharides or polyols to form five- or six-membered cyclic

#### *Improving the Study of Protein Glycosylation with New Tools for Glycopeptide Enrichment DOI: http://dx.doi.org/10.5772/intechopen.97339*

esters. This property has been used to capture glycoproteins and glycopeptides [22]. Importantly, the covalent linkage is reversible at acidic pH which results in release of intact glycopeptides [20]. The interaction between boronic acid and sugars is relatively weak but newly characterized derivatives show promise for enrichment of low-abundance glycopeptides [19]. A final consideration is that boronic acid enrichment does not discriminate between N- and O-linked glycopeptides.
