Advances in mass spectrometry (MS)-based proteomics, including nano liquid chromatography electrospray ionization (ESI) interfaces, faster and more sensitive mass analyzers, and robust bioinformatics approaches, have brought the unbiased discovery of disease biomarkers within reach. These technological improvements have led to a new era of research aimed at improving prognoses, diagnoses, and monitoring responses to therapy through detection of biomarkers in human body fluids (i.e., plasma, saliva and urine). However, making this an effective strategy requires panels of verified disease-specific reporter molecules that, as yet, do not exist. Therefore, there is a tremendous interest in discovery efforts.
Overall, investigators who focus on the early stages of discovery pipelines use two approaches. The first is brute force protein identification to determine differences between samples obtained from patient subjects and control individuals. The inherent complexity of body fluids requires extensive sample separation, usually achieved by a series of orthogonal/complimentary chromatographic steps. Successful studies typically require large amounts of starting material, time, and expertise [1
]. Second, other investigators have used targeted approaches to reduce complexity. One way to direct these experiments is by considering the biology of the disease of interest, in this case, cancer. In this regard, post-translational modifications (PTMs) are especially interesting because they are linked to the disease process and in some cases play a causal role [2
]. Since these modifications can appear at multiple positions on a protein scaffold and on multiple protein backbones, the expression of these motifs is usually greatly amplified as compared to that of single proteins. Thus, through the use of affinity capture reagents, specific PTMs may be exploited as targets to enrich molecules that are a signature of a particular disease state.
Aberrant carbohydrate modifications have been recognized as a hallmark of cancer for over 30 years [3
]. Intriguingly, many of the oldest and most widely used clinical diagnostic tests detect glycoproteins. These include carcinoembryonic antigen (CEA), commonly used as a marker of colorectal cancer; CA-125, frequently employed to diagnose ovarian cancer; and prostate-specific antigen (PSA) [4
]. Interestingly, many of the most informative tests directly assess the expression of a particular class of carbohydrates termed Lewis (Le) blood group antigens, which exhibit unique biological functions [7
]. Anti-sialyl Lea
(CA 19-9), -Lex
, -sialyl Lex
, and -Ley
antibodies are used in the evaluation of biopsy specimens from breast, bladder, colorectal, esophageal and non-small cell lung carcinoma [8
]. In all instances, Le antigen expression is correlated with increased metastasis, advanced stage of disease and reduced survival time. The fact that cancer-related carbohydrate changes are correlated with clinically relevant outcomes such as metastasis and survival enhances their utility as biomarkers. Indeed, studies have already shown that selectively enriching cancer-related protein glycoforms affords the possibility of increasing diagnostic sensitivity and specificity. For example, separation of serum PSA by the Maackia amurensis
agglutinin lectin, which specifically binds α2,3-linked sialic acid, allows discrimination (p < 0.001) between blood samples from individuals with benign prostatic hypertrophy and prostate cancer patients [16
], which standard PSA tests fail to do [17
The relationship between saccharide expression and disease progression has a biological basis as glycans regulate many processes involved in tumorigenesis. For instance, extravasation, a critical step in metastasis, is initiated by shear stress-induced interactions between selectin family endogenous lectins and their cell surface carbohydrate ligands. The selectin family consists of three members with differential expression patterns. Endothelial cells display E- and P-selectin, platelets express P-selectin, and leukocytes present L-selectin [18
]. Carbohydrate ligands for selectins are modified Le blood group antigens, which are abundant in malignant tissues. Evidence is accumulating that the clinical correlation between tumor expression of selectin ligands and metastasis is a reflection of a causal relationship [20
]. Mice lacking L- or P-selectin, singly or in combination, exhibit greater resistance to the metastasis of colon carcinoma cells than do wild-type animals [21
], and E-selectin is also implicated in tumor metastasis [23
]. Additionally, cancer-related protein scaffolds have been identified as selectin ligand carriers, and thus as participants in the extravasation process. For instance, CEA, the glycoprotein commonly used as a marker for colorectal cancer, and CD44, a highly glycosylated cell surface adhesion protein, can serve as E- and L-selectin ligands [20
In light of the strong biological and clinical correlations between tumors and specific types of glycosylation, several laboratories have applied a variety of glycan-based enrichment strategies to cancer biomarker discovery. In general, these studies have tended to be small-scale proof-of-principle endeavors. It is noteworthy that, on the whole, they succeeded in identifying target biomarker candidates for further validation. Thus, the published literature supports the theory that focusing the selection process on cancer-relevant glycans increases the likelihood of identifying potential disease sentinels and/or targets. In terms of specific methods, a variety of protocols, including lectin-, antibody- and chemical-based approaches, have been developed for the capture of glycoproteins and glycopeptides. Lectin-based protocols include those using single species [24
], as well as mixtures, e.g.
, concanavalin A, wheat germ agglutinin, and Artocarpus integrifolia
(jacalin). The latter method has been dubbed multi-lectin affinity chromatography [27
]. Less common are protocols employing carbohydrate-reactive antibodies such as those recognizing Le blood group antigens [31
]. In addition, two similar approaches, involving hydrazide or boronic acid chemistry, capitalize on the cis
-diols in monosaccharides to covalently couple the glycans to a derivatized support [32
Here, we present the results of an extensive method development effort in which we used glycopeptide standards as positive and negative controls to optimize an automated workflow for the rapid enrichment of selectively glycosylated peptides from depleted, trypsin-digested human plasma. Specifically, we performed lectin affinity chromatography at the glycopeptide level. Using a number of requirements, including the presence of an N-linked consensus motif (NXS/T) and a +1 Da mass shift at the modified Asn residue after deglycosylation with PNGase F, we identified glycosites enriched by a specific lectin, an indication that they carried specialized carbohydrate modifications. Using commercially available preparations of Sambucus nigra agglutinin (SNA), which binds sialic acid α2,6- and α2,3-linked to terminal galactose, and Aleuria aurantia lectin (AAL), which binds α-linked fucose at both core and branch positions, we identified a total of 227 glycosites in 119 glycoproteins from human plasma. These included low-abundance proteins such as cadherin-5 and neutrophil gelatinase-associated lipocalin. Now, we are poised to compare families of SNA- and AAL-binding glycoproteins in cancer-relevant human samples.