Acetone precipitation was evaluated as a rapid, simple, low-cost, and efficient method for the selective purification of O-glycopeptides from enzymatic digests of glycoproteins. Ovalbumin (OVA), human and bovine α-acid glycoprotein (hAGP and bAGP), human apolipoprotein C-III (APO-C3), and recombinant human erythropoietin (rhEPO) were used to obtain enzymatic digests with a broad and varied set of peptides, N-glycopeptides, and O-glycopeptides. After digestion and before capillary electrophoresis mass spectrometry (CE-MS) analysis, the amount of ice-cold acetone added to the digests was optimized to maximize recoveries of O-glycopeptides. Furthermore, the different behavior of peptides, N- and O-glycopeptides was explained by studying with multivariate data analysis methods the influence of several physicochemical parameters and properties related to their composition and structure. Principal component analysis (PCA) and, afterward, partial least-squares discriminant analysis (PLS-DA) were used to identify the most significant variables and their importance to differentiate between peptides, N-glycopeptides and O-glycopeptides, or within these classes. This information was useful to understand precipitation of these compounds after addition of acetone and for the selection of the optimal conditions for purification of specific O-glycopeptide biomarkers. Special attention was paid to O-glycopeptide glycoforms of rhEPO because of their applicability in biopharmaceutical quality control and doping analysis.
Complex carbohydrates are ubiquitous in nature and represent
one
of the major classes of biopolymers. They can exhibit highly diverse
structures with multiple branched sites as well as a complex regio-
and stereochemistry. A common way to analytically address this complexity
is liquid chromatography (LC) in combination with mass spectrometry
(MS). However, MS-based detection often does not provide sufficient
information to distinguish glycan isomers. Ion mobility-mass spectrometry
(IM-MS)—a technique that separates ions based on their size,
charge, and shape—has recently shown great potential to solve
this problem by identifying characteristic isomeric glycan features
such as the sialylation and fucosylation pattern. However, while both
LC-MS and IM-MS have clearly proven their individual capabilities
for glycan analysis, attempts to combine both methods into a consistent
workflow are lacking. Here, we close this gap and combine hydrophilic
interaction liquid chromatography (HILIC) with IM-MS to analyze the
glycan structures released from human alpha-1-acid glycoprotein (hAGP).
HILIC separates the crude mixture of highly sialylated multi-antennary
glycans, MS provides information on glycan composition, and IMS is
used to distinguish and quantify α2,6- and α2,3-linked
sialic acid isomers based on characteristic fragments. Further, the
technique can support the assignment of antenna fucosylation. This
feature mapping can confidently assign glycan isomers with multiple
sialic acids within one LC-IM-MS run and is fully compatible with
existing workflows for
N
-glycan analysis.
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