Glycosylation is one of the most common and important post-translational modifications (PTM) found on a protein. The alterations in glycan structures of these proteins are associated with various developmental and pathological states of glycoproteins and have great biological significance. The direct involvement of glycans in biological functions is to confer certain physiochemical properties onto proteins and to act as signals in cell-surface recognition phenomena. Glycoproteins currently constitute an active area of research. Since the functional significance of the glycan moieties have become increasingly apparent, there has been a need for rapid, reliable methods for their determination.
In the area of biotherapeutics, recombinant monoclonal antibodies (mAbs) have gained significant importance in diagnostic and therapeutic applications over the past years. The general structural features of mAbs, such as assembly of light and heavy chains via disulfide bridges, are commonly known. However, the heterogeneity of antibodies, mostly due to the variation of attached sugar moieties, requires thorough characterization. This is necessary to verify the correctness of the overall molecule and provide a reproducible, safe, and effective biological drug compound. Intact glycoprotein profiling is one such approach, in which high-resolution, accurate-mass (HR/AM) mass spectrometers are used to ascertain expected pattern and degree of glycosylation.
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Workflow Overview for Intact Glycoprotein Profiling
Intact glycoprotein profiling by mass spectrometry is typically done on glycoproteins that have been enriched or purified. The enriched protein mixture is introduced into the mass spectrometer using either direct infusion or liquid chromatography coupled to an ESI source. Direct infusion provides more time for signal averaging. For more complex intact-glycoprotein mixtures, on- or off-line LC separation may be required to reduce precursor spectral complexity and minimize ion suppression. Subsequent data analysis identifies the molecular weight and various glycoforms.
Sample Preparation Workflow for Intact Glycoprotein Profiling
Detergents are widely used in glycoprotein chemistry. They aid in solubilization as well as stabilization of glycoprotein complexes. In particular, membrane-bound proteins frequently require detergent treatment in order to dissolve. Unfortunately, detergents are completely unsuitable for ESI-MS and should be avoided whenever possible. It should also be noted that LC does not necessarily solve the problem of detergent interferences with the ionization process. The presence of sodium dodecylsulfate (SDS) should always be avoided, or dramatically reduced where possible.
The glycoproteins are analyzed by MS as intact molecules and thus do not require any digestion.
Mass Spectrometry Workflow for Intact Glycoprotein Profiling
Due to the heterogeneity and variation of the attached glycan moieties, intact glycoprotein profiling is best performed on the hybrid Orbitrap mass spectrometers. These complex samples benefit from the highest available resolution, accurate mass, and fast scan speeds offered by these systems to distinguish the various glycoforms present on a single protein.
Depending upon the complexity of the sample, intact glycoprotein profiling can be performed by either direct infusion or LC-MS. For complex samples where the total number of isoforms might be large, upfront LC separation is required prior to MS analysis. Due to the unique properties of polymer-based monolithic stationary phases, monolithic columns have become an attractive alternative for packed columns, especially for the separation of complex glycoprotein samples. The Thermo Scientific ProSwift RP-10R monolithic column provides robust and efficient separation of glycoproteins and can be directly coupled to the mass spectrometer for online LC-MS analysis.
Data Analysis Workflow for Intact Glycoprotein Profiling
One of the intrinsic advantages of ESI-MS is its ability to place multiple charges on particular species. The multiple charging effect makes the analysis of large intact proteins using conventional mass ranges possible. For these reasons mass spectrometers with the highest available resolution and accurate mass are required to isotopically resolve peaks for precise molecular mass determination. Thermo Scientific Protein Deconvolution software is the only protein deconvolution software available today that takes full advantage of the ultra-high-resolution, accurate-mass data produced by Orbitrap-based mass spectrometers. It employs two different deconvolution algorithms, each optimized for a different type of data, to ensure the highest quality results.
Protein Deconvolution software
- Uses two algorithms, Xtract and ReSpect, which are optimized for isotopically resolved and unresolved data respectively
- Deconvolutes isotopically unresolved data, across a wide mass range of up to 160,000 Da with unsurpassed speed, for compatibility with fast chromatography
- Produces highly accurate results, even for low-abundance proteins, enabling detection of various glycoforms
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