The Analysis of N-linked Glycans by MALDI QIT TOF Mass Spectrometry

User Benefits

- Structural Characterization of glycan PTMs acheived by step-wise MS fragmentation - Easier spectral interpretation due to singly-charged ion species and high resolution, high mass accuracy in all modes of operation - High sensitivity and low chemical noise permits access to fragmentation spectra beyond MS3 on low abundance glycans

Introduction

In recent years enormous research efforts have been devoted to a fuller understanding of the expressed proteome of a wide range of organisms. This has been accelerated by the publication of the genome sequences of human, mouse, rice, Arabidopsis and so on. It is already clear that the dynamic nature of the proteome makes its correlation to cell biology, disease and drug development very challenging. This complexity is added to by the presence of post- translational modifications (PTM). Of these, perhaps the best understood are the phosphorylations which are largely transient and under the discrete control of kinases and phosphorylases. Despite the understandable interest in the phosphorylation state of proteins, a more significant class of PTM exists, namely carbohydrates (or glycans). These are present on more than 50% of eukaryotic proteins and can infer considerable changes in the properties of proteins. They can be split into three classes: N-linked glycan structures which are attached to an asparagine in the amino acid sequence motif Asn-X-Ser/Thr (e.g. fetuin, tPA); O-linked glycan structures coupled to Serine or Threonine residues (e.g. mucins); and GPI anchored proteins which incorporate a central carbohydrate segment linking the protein moiety to an interaction with the cell membrane. Within each of these classes there is enormous potential for heterogeneity since carbohydrates can form branched as well as linear structures and the substitution of one sugar for another e.g. hexoses such as glucose, galactose and mannose. For this reason, here we focus on the N-linked glycans solely. There are many questions that one would want to address with regard to a protein’s glycosylation state, including: Is it glycosylated? What linkages are possible and where according to the amino acid sequence? Which structures are present and where is each one located? Accordingly, a wide number of techniques may need to be applied to deliver the answers and each of these will make a different contribution but might include HPLC, gel electrophoresis, NMR, enzymatic digestion, chemical deglycosylation and mass spectrometry (MS). As the area of proteomics has developed, the key techniques that have emerged are 2D gel electrophoresis and tryptic-digest peptide mass fingerprinting combined with MALDI mass spectrometry. This gives access to gel-based arrays of proteins which can be stained to reveal presence of glycoproteins and these can then be identified and interrogated using the high sensitivity of MALDI mass spectrometry. However, the two overlapping sets of peptide and glycan masses can occlude each other. To overcome this in the case of N-linked glycans, a general enzymatic cleavage can be performed that removes all of the glycans. The enzyme used to achieve this, Protein N-Glycosidase F (PNGase F, Sigma Aldrich Product Code P 7367), results in the deamidation of the Asparagine to an Aspartate and an increase in mass of 1 Da for the associated peptide. It is then possible to compare the glycosylated and deglycosylated mass patterns. This will allow undisturbed peptide mass fingerprinting to identify the protein and then assist in the localization of likely glycosylation sites. Additionally, the PNGase F -released glycans can be recovered and analyzed independently by various methods including further mass spectrometry and targeted deglycosylations to determine the detailed structure. Despite its speed, convenience, simplicity and sensitivity, conventional MALDI-TOF has not had the ability to reveal the detailed structure of N-linked glycans. This is in part due to the fact that most sugars share common residue masses and cannot be identified uniquely in a facile manner. Additionally, determination of the linkage between sugar residues in a glycan requires cross-ring cleavages of the sugars. To achieve this requires MS instrumentation which retains all the features mentioned above but is able to perform step-wise fragmentation of individual precursor ions and ideally through so called MSn technology.

December 6, 2013 GMT