2 research outputs found
Mass Spectrometric Quantification of N‑Linked Glycans by Reference to Exogenous Standards
Environmental
and metabolic processes shape the profile of glycoprotein
glycans expressed by cells, whether in culture, developing tissues,
or mature organisms. Quantitative characterization of glycomic changes
associated with these conditions has been achieved historically by
reductive coupling of oligosaccharides to various fluorophores following
release from glycoprotein and subsequent HPLC or capillary electrophoretic
separation. Such labeling-based approaches provide a robust means
of quantifying glycan amount based on fluorescence yield. Mass spectrometry,
on the other hand, has generally been limited to relative quantification
in which the contribution of the signal intensity for an individual
glycan is expressed as a percent of the signal intensity summed over
the total profile. Relative quantification has been valuable for highlighting
changes in glycan expression between samples; sensitivity is high,
and structural information can be derived by fragmentation. We have
investigated whether MS-based glycomics is amenable to absolute quantification
by referencing signal intensities to well-characterized oligosaccharide
standards. We report the qualification of a set of N-linked oligosaccharide
standards by NMR, HPLC, and MS. We also demonstrate the dynamic range,
sensitivity, and recovery from complex biological matrices for these
standards in their permethylated form. Our results indicate that absolute
quantification for MS-based glycomic analysis is reproducible and
robust utilizing currently available glycan standards
Discrimination between Adenocarcinoma and Normal Pancreatic Ductal Fluid by Proteomic and Glycomic Analysis
Sensitive and specific biomarkers
for pancreatic cancer are currently
unavailable. The high mortality associated with adenocarcinoma of
the pancreatic epithelium justifies the broadest possible search for
new biomarkers that can facilitate early detection or monitor treatment
efficacy. Protein glycosylation is altered in many cancers, leading
many to propose that glycoproteomic changes may provide suitable biomarkers.
In order to assess this possibility for pancreatic cancer, we have
performed an in-depth LC–MS/MS analysis of the proteome and
MS<sup>n</sup>-based characterization of the N-linked glycome of a
small set of pancreatic ductal fluid obtained from normal, pancreatitis,
intraductal papillary mucinous neoplasm (IPMN), and pancreatic adenocarcinoma
patients. Our results identify a set of seven proteins that were consistently
increased in cancer ductal fluid compared to normal (AMYP, PRSS1,
GP2-1, CCDC132, REG1A, REG1B, and REG3A) and one protein that was
consistently decreased (LIPR2). These proteins are all directly or
indirectly associated with the secretory pathway in normal pancreatic
cells. Validation of these changes in abundance by Western blotting
revealed increased REG protein glycoform diversity in cancer. Characterization
of the total N-linked glycome of normal, IPMN, and adenocarcinoma
ductal fluid clustered samples into three discrete groups based on
the prevalence of six dominant glycans. Within each group, the profiles
of less prevalent glycans were able to distinguish normal from cancer
on this small set of samples. Our results emphasize that individual
variation in protein glycosylation must be considered when assessing
the value of a glycoproteomic marker, but also indicate that glycosylation
diversity across human subjects can be reduced to simpler clusters
of individuals whose N-linked glycans share structural features