Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry

Abstract

Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of "unknown" proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL 3 . The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-ð2 → 3Þ-Gal-βð1 → 3Þ-GalNAc, þ656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level.quantitative proteomics | Fourier-transfrom mass spectrometry | electron transfer dissociation | top-down proteomics | posttranslational modifications

Structure of HDL: Particle Subclasses and Molecular Components

“…This is then linked to reduced inhibition of lipoprotein lipase and improved triglyceride clearance. In line, the production rate of apoC-III 1 and -III 2 is more strongly correlated with plasma triglyceride levels than apoC-III 0 (Mauger et al 2006), increased apoC-III 1 / apoC-III 0 ratio has been found in diabetic subjects (Jian et al 2013), and HDL3 from subjects with low HDL-C is characterised by higher levels of monosialylated apoC-III than subjects with high HDL-C (Mazur et al 2010). 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).…”

“…P02656) is generally found as three charge isoforms depending on O-linked glycosylation (GalGalNAc) at Thr94 with or without sialylation; disialylated apoC-III 2 , monosialylated apoCIII 1 and non-sialylated apoC-III 0 (Karlsson et al 2005;Bruneel et al 2008). An early report showed that glycosylation is not necessary for apoC-III secretion and does not affect its relative affinity to different lipoprotein particles (Roghani and Zannis 1988), and, as judged by gel electrophoresis and MS analysis of HDL and plasma, the non-sialylated variant is least abundant, usually less than 5 % of total apoC-III in normal individuals (Wopereis et al 2003;Bruneel et al 2008;Mazur et al 2010;Holleboom et al 2011). In addition to glycosylation, apoC-III can also be C-terminal truncated (des-Ala and des-Ala-Ala), which further increases the number of isoforms (Bondarenko et al 1999;Jin and Manabe 2005;Nicolardi et al 2013a).…”

“…Consequently, such applications in the field of HDL are being presented by using, e.g. multiple reaction monitoring and top-down proteomics with high-resolution MS (Mazur et al 2010;Sung et al 2012). Another interesting and fairly simple approach is to use ratio determinations, e.g.…”

Top-down proteomics: applications, recent developments and perspectives

“…The authors reported reproducible <10 ppm mass accuracy with this instrument on intact proteins and confidently identified the proteins using CID fragmentation (MS2 and MS3). The study of low molecular weight proteins from human blood including the quantitation of apolipoprotein proteoforms [79,80] using LTQ-orbitrap instrumentation. The LTQ-orbitrap was also used to distinguish several glycoforms from intact recombinant antibodies (150 kDa) and fragment the reduced light and heavy chains using CID [81].…”

Epidemiology: Disease Associations and Modulators of HDL-Related Biomarkers

“…Proteomics as a new promising approach for detecting biomarkers and mechanisms could potentially explain the beneficial characteristics of HDL particles unrelated to their cholesterol content. To this end, novel sophisticated methods have been recently developed (Gordon et al 2010;Mazur et al 2010;Burillo et al 2013;Hoofnagle et al 2012;Mazur and Cardasis 2013;Riwanto et al 2013). High-throughput methods are urgently needed for large-scale epidemiological studies.…”