Identification of New Apolipoprotein-CIII Glycoforms with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry of Human Sera

Abstract

Apolipoprotein-CIII (apoCIII) is an abundant blood glycoprotein associated with lipoprotein particles. Three different glycoforms have been described, all containing a mucin-type core-1 O-glycosylation with either zero, one or two sialic acids. Changes in the relative abundance of these glycoforms have been observed in a variety of different pathologies. In this study, ultrahigh resolution 15T MALDI Fourier transform ion cyclotron resonance (FTICR) MS was used to analyze apoCIII isoforms in serum protein profiles. For this purpose, serum proteins were purified using both a fully automated RPC18-based magnetic bead method and an RPC4 cartridge-based solid phase extraction method. Six new apoCIII isoforms were identified with low-ppm mass measurement errors and ultrahigh precision. These were characterized by more complex glycan moieties that are fucosylated instead of sialylated. To confirm the glycan moiety and localize the glycosylation site, top-down ESI-FTICR-MS/MS and bottom-up LC-ion trap MS/MS were used. A large variation in the presence and abundance of the fucosylated isoforms was found in a set of 96 serum samples. These findings of fucosylated apolipoprotein-CIII isoforms warrant further research to elucidate the implications these glycoforms may have for the plethora of studies where alterations in apoCIII have been linked to the development of many different pathologies.

Structure of HDL: Particle Subclasses and Molecular Components

“…The evaluation of apoC-III isoforms is complicated by the fact that apoC-III 0 can be separated into a non-glycosylated form and glycosylated but non-sialylated forms (Bruneel et al 2008;Holleboom et al 2011;Nicolardi et al 2013a). Moreover, a recent MS study of 96 serum samples showed that 30 % of the individuals displayed an apoC-III pattern with additional glycosylated variants, characterised by fucosylation (Nicolardi et al 2013b). The relevance of these glycosylated non-sialylated variants of apoC-III, as of the C-terminal truncated forms, is yet unclear, but may explain somewhat contradictory results showing higher relative levels of non-and lesssialylated apoC-III in obese subjects than in lean subjects (Harvey et al 2009;Karlsson et al 2009), although obesity is generally associated with high triglyceride levels.…”

SPE-MALDI Profiling of Serum Peptides and Proteins by Ultrahigh Resolution FTICR-MS

“…These steps include a baseline correction, smoothing, peak-picking, internal calibration or peak alignment, and transformation of the data in a format compatible with methods for statistical analysis. This data processing becomes more robust with improved quality of the profiles, and moreover, high quality data lead to improved statistical analysis [13]. In this context, resolving power, dynamic range, mass measurement accuracy, and precision of the mass analyser are parameters that define the quality of the signature.…”

“…Additional MS/MS-experiments can be performed on the FTICR itself (in electrospray mode), or using other MS-equipment. For example, we have recently reported on the identification of new apolipoprotein CIII glycoforms in MALDI-FTICR profiles that were recorded up to an m/z-value of 10,900 [13]. With the current extension of the m/z-range up to 15 kDa, these MALDI-FTICR profiles can be used for both bottomup and top-down identifications [14,15].…”

MALDI Profiling and Applications in Medicine

“…Other studies have focussed upon different glycosylation forms of specifi c proteins of interest. The application of high accurate mass MALDI profi ling after purifi cation allowed for the identifi cation of six new apolipoprotein CIII isoforms and their glycosylation status which may have a role in lipid metabolism and transport within the body and ApoCIII isoforms have been identifi ed as easy to purify by microscale tip preparation and able to allow for diagnoses in chronic hepatitis C and alcoholic liver cirrhosis [ 61 ]. A different approach sought to study the glycosylation status of membrane-type 1 matrix metalloproteinase (MT1-MMP) via MALDI mass spectrometric analysis after immunoprecipitation from cell lines as the glycosylation is required for cancer cell invasive processes [ 81 ].…”