Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis

Abstract

Glycosylation is a common post-translational modification that can add complexity to the proteome of many cell types. We used enzymatic and chemical methods of deglycosylation to treat a heavily glycosylated exoproteome sample from the filamentous fungus Trichoderma reesei. Deglycosylated samples were resolved on one-dimensional (1-D) and two-dimensional (2-D) gels in order to determine the effect of deglycosylation on the electrophoresis patterns and on the ability to identify proteins by peptide mass matching using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of in-gel tryptic digests. We found that deglycosylation of the protein sample resulted in different protein patterns on 1-D and 2-D gels, reduced the complexity of gel patterns, and enhanced the protein identification of some proteins via MALDI-TOF-MS. Deglycosylation with trifluoromethanesulfonic acid (TFMS) was found to be more effective than enzymatic treatments. These deglycosylation techniques may be employed in whole proteome analysis to locate glycosylated proteins and assist in their identification by MS.

Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus

“…First, the majority of the proteins secreted by filamentous fungi can have carbohydrate contents reaching up to 50% of the total molecular weight of the protein (Peberdy, 1994). This heavy glycosylation is thought to be responsible for the tendency of these proteins to show ''smearing'' on SDS-PAGE gels (Fryksdale et al, 2002). Second, the concentration of glycosylated proteins entering the first-dimensional gel (the IPG strip) may be low (Packer and Keatinge, 2001) and good mass spectra may not be obtained from low quantities of protein.…”

“…However, as the 2-DE pattern of rutin-induced and non-induced proteins did not seem to be affected by enzymatic deglycosylation, it can be assumed that the isoforms were not necessarily due to glycosylation differences, but to other types of post-translational modifications such as sialylation (L€ oster and Kannicht, 2002). Fourth, in some cases, glycosylation protects proteins against proteolysis, making the identification of the protein more difficult by peptide mass matching as this technique relies on efficient protease digestion prior to MS (Fryksdale et al, 2002). Lastly, N-linked oligosaccharides can be difficult to see by MALDI-TOF MS because they tend to make the mass of the tryptically digested peptides very large (above 3500 Da), and thus appear as broad band signals in the mass spectra (Packer and Keatinge, 2001).…”

Protein stains for proteomic applications:Which, when, why?

“…Deglycosylation of the protein sample, with the above enzymatic treatments or with trifluoromethanesulfonic acid, reduces the complexity of gel patterns on 1-DE and 2-DE gels, and enhances the protein identification of some proteins via MALDI-TOF-MS [171]. Protocols for in-gel deglycosylation prior to MS analysis with reference to standard patterns with untreated samples have been optimized, for the identification of the (apo)proteins, for the recognition of the glycosylation sites [172] and for the characterization of the glycan moieties [173,174].…”

The Use of Mass Spectrometry for Characterization of Fungal Secretomes

“…This matter is very difficult to confront since the complexity of multienzyme systems is already difficult. Nevertheless, post translational modifications have been detected in cellulases of Trichoderma reesei (Fryksdale et al 2002). In this study the enzymes were deglycosylated previous to two-dimensional electrophoresis; this made changes in protein pattern and also improved protein identification by mass spectrometry, because large glycosylation block trypsin.…”