Novel mass spectrometric immunoassays for the rapid structural characterization of plasma apolipoproteins

Abstract

Novel mass spectrometric immunoassays (MSIAs) for the isolation and structural characterization of plasma apolipoprotein A-I (apoA-I), apoA-II, and apoE have been developed. The assays combine selective isolation of apolipoprotein species via affinity capture with mass-specific detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In application, plasma (from 50 l of whole blood drawn from individuals, using finger lancet) was addressed with affinity pipette tips derivatized with antibodies toward the specific apolipoprotein. The time required for each assay was ‫ف‬ 15 min, less if assays on multiple individuals were performed in parallel. In a brief study of five individuals, several recently reported apoA-II variants were identified and observed consistently in all individuals. Additionally, the apoE phenotype of E3/ E3 was observed in three of the individuals, and E2/E3 and E3/E4 observed in the remaining two individuals, the latter of whom suffers from Alzheimer's disease. Overall, the MSIA approach offers a rapid, sensitive, and highly accurate means of profiling apolipoproteins from small volumes of plasma. The apolipoproteins represent a significant class of proteins whose functions range from enzyme activation (e.g., LCAT and lipoprotein lipase) to lipid/cholesterol transport and clearance. Although well studied over the past two decades, it has only recently become clear that mutations present within the apolipoproteins may be responsible for, or assist in, the progression of disease. Table 1 gives a brief overview of some diseases correlated with specific mutations present within apolipoproteins. Universally, these mutations are commonly studied on the DNA level using a variety of genotyping assays. For instance, apolipoprotein E (apoE) genotyping usually involves PCR amplification of short subsequences of exons 2 and 3 (containing both polymorphic sites) and analysis of the PCR products by: 1 ) dot blot hybridization with allele-specific oligonucleotide probes (1); 2 ) oligonucleotide ligation assay (2); 3 ) single-stranded conformational polymorphisms (3); or, 4 ) restriction fragment-length polymorphism analysis using restriction enzymes recognizing the sites of polymorphism (4). More advanced approaches involve competitive amplified single mutation detection by selective probe hybridization immunoassay (5) or primer-extension mass spectrometry assays directed at the polymorphic sites within the gene (6). This latter assay brings to the analysis the direct detection of indicators (extended primers containing the base complement of the polymorphism) at exact mass values, thus enabling an assay that is free of artifacts encountered in other assays using, e.g., electrophoretic gel shifts or differential hybridization.As with the nucleic acid mass spectrometry assays, direct mass spectrometric analysis of proteins (or proteolytic fragments) brings an unprecedented degree of analytical accuracy to the analysis of point mutations. However, whereas the polym...

Identification of Apolipoprotein A-II in Cerebrospinal Fluid of Pediatric Brain Tumor Patients by Protein Expression Profiling

“…The masses of the 2 protein peaks (m/z 17 248 and 17 369) in the m/z 17 000 protein peak cluster in our SELDI spectra were in concordance with previously described masses of isoforms of APO A-II (25,26 ). Multiple isoforms of APO A-II have been described in humans (25,(27)(28)(29) and are the result of posttranslational modifications, such as oxidization or truncation of the C-terminal glutamine residue (25)(26)(27)(28).…”

Glycosylation status of vitamin D binding protein in cancer patients

“…Since its inception in 1995 by R.W. Nelson et al, 21 it has been applied to study a variety of proteins and peptides, [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] and has served as the foundation for the nascent field of Population Proteomics. [37][38][39][40][41][42] Roughly, MSIA can be envisioned as ultra high resolution, semiquantitative Western blotting in which protein variants are thoroughly resolved and their molecular masses determined to within less than 2 Da.…”

“…Isolation of DBP from plasma was carried out with proprietary MSIA pipette tips from Intrinsic Bioprobes (Tempe, AZ) derivatized with the DBP antibodies via 1,1 0 -carbonyldiimidazole (CDI) chemistry as previously described. 33,43 Serum samples from 29 cancer patients (nine breast cancer, 11 colorectal cancer, nine prostate cancer) of African-American, Caucasian, and Hispanic origin, along with age, gender, and racematched controls were purchased from ProMedDx (Norton, MA). Twenty seven plasma samples from pancreatic cancer patients were generously donated by Dr. Douglas Lake of the School of Life Sciences at Arizona State University.…”

“…With the aid of a Beckman Multimek 96-channel automated pipetter, MSIA pipette tips that had been preactivated with CDI 33,43 were derivatized with polyclonal DBP antibodies by repetitively flowing (aspirating and dispensing 750 times) 50 lL volumes of antibody solution (150 lL/well; 0.05 g/L in 0.1M MES buffered saline, pH 4.7) through the tips. Antibody-linked tips were stored in HBS at 4 C until the day of use at which time they were prerinsed (400 lL/well; 150 lL aspirate and dispense cycles; 10 cycles) with HBS then used to extract DBP from individual samples (25 lL of human serum or plasma diluted with 75 lL of HBS) at room temperature (100 lL/well; 50 lL aspirate and dispense cycles; 750 cycles).…”

Full-Length Characterization of Proteins in Human Populations

“…Work over the past 2 decades has led to the development of high-throughput mass spectrometric immunoassay (MSIA) 2 technologies and workflows that are well suited to the standardized characterization of protein diversity in human populations (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Data from such "population proteomics" studies include changes in concentration, amino acid sequence, and relative qualitative/quantitative changes in posttranslational modifications (15,16 ).…”