What Has Mass Spectrometry Ever Done for Us?

Mass spectrometry is one of Peak Proteins’ core services. Using the Sciex ExionLC coupled to a Sciex X500B QTOF mass spectrometer with Sciex OS and BioPharmaView Software, we have the ability to perform both LC-MS intact protein analysis along with LC-MSMS peptide mapping analysis.

Our new recruit Richard Mott was having a chat with our resident mass spectrometry expert, Rachel Rowlinson, the other day on Zoom (tea and cake included).  Reminiscent of the classic Monty Python sketch “What Did the Romans Ever Do for Us” from the Life of Brian (for those who are intrigued or feeling nostalgic have a search on YouTube), the conversation went something like this:

Richard: So, Rachel what has Mass Spectrometry ever done for us?

Rachel: Well there are so many examples but the most common is determining the intact mass of proteins. It is very much a routine measurement for us. What we are looking for is to confirm identity of the protein to show that we have made what we think we have made. Often that is after a reaction, such as biotinylation or proteolytic de-tagging, that we have performed as part of a purification procedure. In addition, we are looking for any post translational modifications or truncations that might have occurred on our protein of interest.

Richard: What happens if you find any modifications? Are you able to look into that in any more detail?

Rachel: Yes absolutely, we often digest a protein with a protease, usually trypsin. We then inject the whole mixture onto the mass spectrometer via the inline Reverse phase HPLC (high pressure liquid chromatography) column performing LC-MSMS. This generates a peptide map which is basically the masses of all of the individual peptides along with related sequence masses. From that, by comparing the data to what is predicted from the full amino acid sequence of the protein we can tell exactly where in the sequence any modification or truncation has occurred.

Richard: That sounds pretty useful, what are the main applications of that technique that help our customers with their individual projects?

Rachel: Well many of our customers are studying kinases and other proteins that require knowledge of the phosphorylation status of the purified protein. They can use the intact mass data to show that yes their protein is phosphorylated and how many times and determine if they have a mixture of phosphorylated species present in the sample. Following on from that they can go further and show which sites are phosphorylated using the peptide mapping methods.

Richard: Oh I see – Brilliant – anything else…?

Rachel: Our customers who are developing biopharmaceutical production processes need to build up a profile of their critical quality attributes (CQAs). For them we have used mass spectrometry data to show what product and process related modifications have occurred on their molecule. Examples include elucidating acetylation sites, truncations, oxidation. Often of equal importance to them is showing what CQAs are not present in their product. Again, we have used both intact mass and peptide mapping in these cases.

Richard: What about disulphide bonds that’s often a CQA, are you able to investigate them with mass spectrometry?

Rachel: Yes, that is especially relevant to customers’ proteins that have been refolded from E.coli inclusion bodies. We use the same peptide mapping technique to show which disulphide bonds are formed and between which disulphides. This often involves choosing a combination of proteases to do the initial digest because you need to generate peptides that are both not too big and yet have the predicted disulphide bond pairing between them. Customers often want to know if there are any mis-formed disulphides. They are usually in low abundance in the sample, yet we can still pick them out if they are present.

Richard: If had a protein sample and I didn’t know what it was, say an impurity from my purification process, are you able to tell me what it actually is?

Rachel: Again yes, we are often asked to find the identity of proteins in an unknown sample. This can be samples such as a band on a gel or a minor peak from a reverse phase HPLC run. Often these samples contain more than one protein and we can still identify all of the components by digesting the sample with a protease (again usually trypsin) and then challenging a database like MASCOT with the mass data from all of the fragments we have generated. The software is incredibly sophisticated and matches peptide fragment masses with known sequences, generating a list of proteins that your sample contains. We had one really interesting project where we identified novel peptide inhibitors of snake venom.

Richard: What about glycosylated proteins? Can you use mass spectrometry to tell anything about where the sugars are attached and what the sugars are?

Rachel: With N linked glycosylation you can look through the sequence of your protein to see if it contains any potential sites (Asn-X-Ser/Thr). That doesn’t mean your protein is glycosylated so you can confirm if it is or not with peptide mapping. O linked glycosylation is a bit more tricky in that there is no particular canonical sequence. For this you have to use a bioinformatics prediction tool such as NETOGlyc 4.0 to find sites in your protein where it is likely to occur. You can then base your peptide mapping strategy on this knowledge to confirm if O linked glycosylation has occurred. We had a project working on the inhibitory receptor G6b-B where mapping the O linked glycosylation was key to generating mutants that we could crystallise and ultimately determine the crystal structure of G6b-B to 2Å (PDB:6ROX).

Richard: I’ve heard the term “Native Mass Spectrometry”, what exactly is that?

Rachel:  It refers to when you are running mass spectrometry experiments under physiological conditions. The beauty of this is that it allows you to obtain mass data on proteins complexed with ligands, or bound to other proteins.  An example of where we have used native mass spectrometry was to study GDP (guanosine diphosphate) binding to proteins of interest to us. It is a relatively new technique and there is a lot of excitement in the field as it is proving to be a really powerful way to study proteins.

Richard: Wow, Rachel, I feel like we have only just scratched the surface of “what mass spec can do for us”.  Thank you so much for sharing some of your insights and experience. It’s a shame we are on a video call or you could have shared some of that lovely looking carrot cake too.