Here we describe novel application areas in which hydrophilic interaction liquid chromatography (HILIC) has vast importance and utility, including peptides, glycopeptides, two-dimensional liquid chromatography in proteomics, oligonucleotides, lipids and lipidomics, glycan analysis, proteins, glycoproteins, phosphoproteins, and related biopolymers. We also describe some specific HILIC conditions that have proven useful for these classes of biologically active analytes and provide typical chromatograms from the recent literature, both scientific and commercial.
In part I of this two-part series on modern hydrophilic interaction liquid chromatography (HILIC) applications of relevance to the biotechnology industry, we discussed the fundamental principles and mechanisms suggested from the literature for this, now very widely accepted and practiced form of modern high performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC). We also discussed just a few of the common applications already described for simple and complex glycans often derived from glycopeptides or glycoproteins or for synthetic, standard glycans. We discussed the current roles of HILIC in glycoprofiling and glycan analysis from any source, but in particular by deglycosylation of proteins and peptides. In part II, we discuss modern usage and specific applications of HILIC for two other major classes of analytes important in biotechnology today: glycopeptides and peptides, and glycoproteins and proteins. Other types of proteins, such as phosphoproteins and their applications, are also discussed. Finally, we propose some possible future applications in which HILIC can prove itself useful, including complex glycans, glycomics, oligo-drugs (for example, heparins), glycosaminoglycans, and others.
PeptidesHILIC stationary phases were developed with the aim of providing a tool for the analysis of hydrophilic compounds, which are poorly retained in reversed-phase liquid chromatography (LC) and, therefore, are not ideally resolved (1). Although the elution order of analytes in HILIC is generally reversed compared to that in reversed-phase LC (that is, the most hydrophobic compounds are eluted first in HILIC and the most hydrophilic are eluted last), it has been recognized that the retention order is not inversely proportional (2). Because of various interactions in the HILIC mode that contribute to the separation and selectivity in reversed-phase LC and HILIC, the techniques to a large degree are orthogonal (3,4).
Besides the analysis of small molecules, many early HILIC applications investigated the separation of peptides. Some of these include those of Alpert (1), Yoshida (5), and Mant and colleagues (6). More recently, Gilar and Jaworski (3) compared three HILIC stationary phases and concluded that for the separation of charged species, such as peptides, the interactions with the charged silanols on silica significantly contributed to the retention mechanism.
The separation orthogonality of HILIC is often used for the analysis of compounds that are difficult to resolve in reversed-phase LC. For example, Zhu and colleagues (2) have used both reversed-phase LC and HILIC for analyses of peptide maps of erythropoietin and found that the methods were complementary. HILIC was more suitable for the detection and quantification of small or hydrophilic peptides and also glycosylated peptides that were not adequately resolved in reversed-phase LC. Moreover, HILIC peptide maps contained some unique, very hydrophobic peptides that were not detected in reversed-phase LC.
HILIC was also applied to the quantitative bioanalysis of therapeutic peptides and proteins. This class of biological therapeutics holds great potential; however, the analysis has been challenging. Biological fluids contain many peptides, especially when the analysts perform proteolysis of serum proteins before liquid chromatography–mass spectrometry (LC–MS). Two-dimensional liquid chromatography (2D LC), reversed phase × HILIC, combined with multiple reaction monitoring mass spectrometry (MS), was used for the quantitation of peptides of interest. Limits of detection below 10 ng/mL were achieved (7).
As stated above, HILIC and reversed-phase LC separation modes are orthogonal, and they can be utilized for 2D LC. D'Attoma and colleagues (8) adopted HILIC as the second dimension of 2D LC for the separation of protein digests. They reported an effective peak capacity of 2600 in a 180-min analysis. A HILIC × reversed-phase 2D LC setup was also evaluated by Di Palma and colleagues (10) for the separation of complex proteomic samples. The authors observed a favorable separation of peptides on zwitterionic HILIC in the first dimension compared to the more typical, first dimension — strong cation-exchange chromatography. Importantly, the authors noted a potential for enrichment of phosphopeptides in HILIC × reversed-phase 2D LC (9,10). McNulty and Annan (11) also investigated the potential of HILIC × reversed-phase 2D LC for proteomic analysis (11). Similar to previous reports, they observed significant advantages of HILIC for phosphoproteomic analysis. Because of charge–charge interactions with the zwitterionic stationary phase, or simply because of the more hydrophilic nature caused by phosphate groups, the phosphopeptides were eluted in a later elution time window when compared to general peptides (9–11). Phosphopeptides also appeared to be better recovered and more detectable in the HILIC × reversed-phase 2D LC method (11). A similar observation was made by Alpert (12), who proposed that the solute's interaction with the charged, hydrophilic sorbent may affect their elution patterns. This separation mode was then termed electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and it can be used for the selective isolation of phosphopeptides from a protein digest. To further this observation, Sze (13,14) has used ERLIC–weak anion-exchange approaches (HILIC on weak anion-exchange surfaces) for selectively retaining phosphopeptides, free of other, acidic peptides, by operating at pH 2, where the phosphate group retains a negative charge and peptide carboxyls are neutral.