The glycoproteome is one of the major subproteomes of human plasma, as many proteins are secreted from the tissues, such as the liver, in a glycosylated form [1—4]. It is proposed from literature studies that about 50% of all plasma proteins are glycosylated , which was confirmed by lectin capture experiments, if one excludes albumin . The plasma glycoproteome has important clinical value, as many bio-markers are glycosylated, such as the breast cancer biomarkers CA125 and ERBB [7,
* Originally published in Proteomics 2005, 13, 3353-3366
8]. Zhang et al.  have developed a method to specifically enrich glycoproteins from human serum by capturing N-linked glycoproteins using hydrazide chemistry. In this method, the captured proteins were digested and the N-linked glycopeptides were then isolated from the complex. The glycopeptides were treated with PNGase F (to release glycans) and identified using MS/MS. This method could also be used for comparative quantification if coupled with isotope labeling. Using lectin affinity to enrich glycoproteins is another approach, which avoids chemical derivatization and the potential for side reactions. In order to comprehensively study the serum glyco-proteome, we have developed a multilectin affinity system to efficiently and specifically enrich glycoproteins from human serum . In that study we demonstrated that the use of a set of lectins (optimized to a given sample) overcame the broad specificity and lack of complete glycoprotein capture that is typically achieved with a single lectin. In addition, we demonstrated that this approach was specific to glycoproteins, gave good recovery and was reproducible.
The challenge of a comprehensive study of the human plasma proteome is its wide dynamic range. To better identify low-abundance proteins in plasma, the removal of the most abundant protein(s) using an immunoaffinity approach has been proven to be effective . However, with the depletion of these high-abundance proteins, such as albumin, it has been suggested that some interesting proteins are also lost due to protein complex formation. The glycoprotein enrichment process described here automatically improves the dynamic range of serum protein analysis, since the nonglycosylated albumin is largely removed. Although some albumin is retained in the affinity systems with its associated glycoproteins, this approach minimizes nonspecific losses.
In this research, we analyzed HUPO human plasma and serum samples from different ethnic groups using multilectin affinity chromatography followed by trypsin digestion and LC-MS/MS. The results of a comparison of the plasma and serum glycoproteomes are reported here, as well as the results obtained with samples collected from different ethnic groups.
Was this article helpful?