Protein Characterization
Post-translational modifications (PTMs)
Analysis of post-translationally modified proteins by mass spectrometry allows for localization of modification sites and, in the case of glycosylation, even structural elucidation of the modification itself. The workflow for PTM analysis is fundamentally the same as for protein identification; the protein is digested by the most suitable enzyme to proteolytic peptides and these are analyzed by nano LC/MS/MS using an LTQ Orbitrap XL mass spectrometer. Product ion data are searched against protein databases and candidate modified peptides are manually validated. A separate analysis of the raw data allows us to see any modified peptides missed by the database search. The ability to measure both parent and product ions at high resolution and mass accuracy allows for high confidence in site assignment. All of our services are supported fully by technical support staff.
Some of the most common PTM services we offer are:
Phosphorylation
We are able to detect and even quantify (for more information click here) the levels of phosphorylation in both in vitro systems or as part of a broad global phosphorylation mapping study.
Glycosylation
Mapping the site of both N- and O-linked glycosylation along with structural elucidation of the glycan itself is a challenging endeavor. NextGen Sciences has gained a wealth of experience in this field over many years and we are able to deliver in-depth analysis with fast turnaround times to make this a viable service versus a research project. The labile nature of the glycan when subjected to MS/MS means multiple analyses (including targeted MS/MS/MS) may be necessary to determine the modification site along with glycan structure.
Intact mass determination
NextGen offers an electrospray-based service for measurement of intact molecular weight for peptides, recombinant proteins, protein drugs and monoclonal antibodies. This may utilize direct infusion or liquid chromatography, dependent on sample complexity, protein abundance and buffer system employed. Typical mass measurement is within 0.01%.
Example MS spectrum showing the protein charge envelope (protein MW is 60,013 Da):
De novo sequencing
De novo sequencing services are available when the source organism is poorly represented and distantly related to any other organism in the protein and EST databases. We also utilize de novo sequencing when working with novel proteins, endogenous peptides (such as naturally occurring peptides) or protein therapeutics.
An example of de novo sequencing is shown for a class I MHC peptide:
