Combined analysis of cleavage sites and glycosylation in a complex mixture

In one of our complex datasets in which we analysed 30 human serum samples using ProQuant™ we noticed that the serum protein hemopexin was found with a range of residues lost from its N-terminus. We detected and quantified the following peptides:

• Hex(1)HexNAc(1)NeuAc(1)
• Hex(1)HexNAc(1)NeuAc(2)

The following peptides were found glycosylated: 

Some peptides have two separate glycosylations, as denoted by asterisks. While the software we use determined a location for the glycosylations, careful investigation of the MS2 fragmentation patterns revealed that the locations of the glycosylations were ambiguous, with multiple potential O-linked glycosylation sites (e.g. Thr-1, Thr-6, Ser-7, Thr-17 and Thr-24 on the longest peptide). We reflect that by showing peptides that contain one or more glycosylations but not their location.

Identities of all the above peptides were checked manually using our purpose-built suite of tools.

The precision with which the abundance of individual peptides can be determined using ProQuant™ means that we can investigate potential interesting biology associated with our observations. We started by determining whether there was a relationship between residues being lost at N-terminus and the O-glycosylation. We quantified the proportion of the protein that has had residues lost from its N-terminus, and looked for the potential effect of glycosylation of the region. We can see clearly that this loss of residues is much more frequent in aglycosylated protein than it is on the glycosylated form. 

What is more, the precision with which we have determined the peptide abundances using ProQuant™ enables us to correlate the fractions of N-terminal residue loss between glycosylated and aglycosylated proteoforms, despite the big differences in those fractions. We observe a strong positive correlation, implying a biological role for this phenomenon.

We are not yet able to share the details, but we have compared the fraction of protein that has lost residues at its N-terminal with a huge range of biochemical, immunological, medical and biomarker data from the same subjects. There is a potentially exciting association between the loss of residues at the N-terminal of hemopexin and both a medical phenotype and other proteomics data, which has opened up a whole new field of research for us.

This technical note highlights a number of key features of ProQuant™. Firstly, observations such as these cannot be obtained without the incredible underlying precision inherent in our data at the peptide level. Secondly, that data has to be interrogated in a way that is open to identifying unusual patterns. Outputting a list of proteins with their relative abundances in the samples is simply insufficient in a world in which the importance of proteoforms is apparent and obvious. The ProQuant™ team here at RxCelerate have a background in the research and investigation into proteoforms carried out as part of Methuselah Health, where we investigated the role of proteoforms in ageing and ageing-related diseases. When you are interested in finding out as much as possible about the proteins and proteoforms in your sample, speak to the experts!