Relationship of apolipoprotein C-III proteoform composition with ankle-brachial index and peripheral artery disease in the Multi-Ethnic Study of Atherosclerosis (MESA).

BACKGROUND AND AIMS: Apolipoprotein C-III (apoC-III) proteoform composition shows distinct relationships with plasma lipids and cardiovascular risk. The present study tested whether apoC-III proteoforms are associated with risk of peripheral artery disease (PAD). METHODS: ApoC-III proteoforms, i.e., native (C-III0a), and glycosylated with zero (C-III0b), one (C-III1) or two (C-III2) sialic acids, were measured by mass spectrometry immunoassay on 5,734 Multi-Ethnic Study of Atherosclerosis participants who were subsequently followed for clinical PAD over 17 years. Ankle-brachial index (ABI) was also assessed at baseline and then 3 and 10 years later in 4,830 participants. RESULTS: Higher baseline C-III0b/C-III1 and lower baseline C-III2/C-III1 were associated with slower decline in ABI (follow-up adjusted for baseline) over time, independently of cardiometabolic risk factors, and plasma triglycerides and HDL cholesterol levels (estimated difference per 1 SD was 0.31 % for both, p < 0.01). The associations between C-III2/C-III1 and changes in ABI were stronger in men (-1.21 % vs. -0.27 % in women), and in Black and Chinese participants (-0.83 % and -0.86 % vs. 0.12 % in White). Higher C-III0b/C-III1 was associated with a trend for lower risk of PAD (HR = 0.84 [95%CI: 0.67-1.04]) that became stronger after excluding participants on lipid-lowering medications (0.73 [95%CI: 0.57-0.94]). Neither change in ABI nor clinical PAD was related to total apoC-III levels. CONCLUSIONS: We found associations of apoC-III proteoform composition with changes in ABI that were independent of other risk factors, including plasma lipids. Our data further support unique properties of apoC-III proteoforms in modulating vascular health that go beyond total apoC-III levels.

Association of apolipoproteins C-I and C-II truncations with coronary heart disease and progression of coronary artery calcium: Multi-Ethnic Study of Atherosclerosis.

BACKGROUND AND AIMS: Higher truncated-to-native proteoform ratios of apolipoproteins (apo) C-I (C-I'/C-I) and C-II (C-II'/C-II) are associated with less atherogenic lipid profiles. We examined prospective relationships of C-I'/C-II and C-II'/C-II with coronary heart disease (CHD) and coronary artery calcium (CAC). METHODS: ApoC-I and apoC-II proteoforms were measured by mass spectrometry immunoassay in 5790 MESA baseline plasma samples. CHD events (myocardial infarction, resuscitated cardiac arrest, fatal CHD, n = 434) were evaluated for up to 17 years. CAC was measured 1-4 times over 10 years for incident CAC (if baseline CAC = 0), and changes (follow-up adjusted for baseline) in CAC score and density (if baseline CAC>0). RESULTS: C-II'/C-II was inversely associated with CHD (n = 434 events) after adjusting for non-lipid cardiovascular risk factors (Hazard ratio: 0.89 [95% CI: 0.81-0.98] per SD), however, the association was attenuated after further adjustment for HDL levels (0.93 [0.83-1.03]). There was no association between C-I'/C-I and CHD (0.98 [0.88-1.08]). C-II'/C-II was positively associated with changes in CAC score (3.4% [95%CI: 0.6, 6.3]) and density (6.3% [0.3, 4.2]), while C-I'/C-I was inversely associated with incident CAC (Risk ratio: 0.89 [95% CI: 0.81, 0.98]) in fully adjusted models that included plasma lipids. Total apoC-I and apoC-II concentrations were not associated with CHD, incident CAC or change in CAC score. CONCLUSIONS: Increased apoC-II truncation was associated with reduced CHD, possibly explained by differences in lipid metabolism. Increased apoC-I and apoC-II truncations were also associated with less CAC progression and/or development of denser coronary plaques.

Apo CIII Proteoforms, Plasma Lipids, and Cardiovascular Risk in MESA.

BACKGROUND: Apo CIII (apolipoprotein CIII) is an important regulator of triglyceride metabolism and was associated with cardiovascular risk in several cohorts. It is present in 4 major proteoforms, a native peptide (CIII0a), and glycosylated proteoforms with zero (CIII0b), 1 (CIII1, most abundant), or 2 (CIII2) sialic acids, which may differentially modify lipoprotein metabolism. We studied the relationships of these proteoforms with plasma lipids and cardiovascular risk. METHODS: Apo CIII proteoforms were measured by mass spectrometry immunoassay in baseline plasma samples of 5791 participants of Multi-Ethnic Study of Atherosclerosis, an observational community-based cohort. Standard plasma lipids were collected for up to 16 years and cardiovascular events (myocardial infarction, resuscitated cardiac arrest, or stroke) were adjudicated for up to 17 years. RESULTS: Apo CIII proteoform composition differed by age, sex, race and ethnicity, body mass index, and fasting glucose. Notably, CIII1 was lower in older participants, men and Black and Chinese (versus White) participants, and higher in obesity and diabetes. In contrast, CIII2 was higher in older participants, men, Black, and Chinese persons, and lower in Hispanic individuals and obesity. Higher CIII2 to CIII1 ratio (CIII2/III1) was associated with lower triglycerides and higher HDL (high-density lipoprotein) in cross-sectional and longitudinal models, independently of clinical and demographic risk factors and total apo CIII. The associations of CIII0a/III1 and CIII0b/III1 with plasma lipids were weaker and varied through cross-sectional and longitudinal analyses. Total apo CIII and CIII2/III1 were positively associated with cardiovascular disease risk (n=669 events, hazard ratios, 1.14 [95% CI, 1.04-1.25] and 1.21 [1.11-1.31], respectively); however, the associations were attenuated after adjustment for clinical and demographic characteristics (1.07 [0.98-1.16]; 1.07 [0.97-1.17]). In contrast, CIII0b/III1 was inversely associated with cardiovascular disease risk even after full adjustment including plasma lipids (0.86 [0.79-0.93]). CONCLUSIONS: Our data indicate differences in clinical and demographic relationships of apo CIII proteoforms, and highlight the importance of apo CIII proteoform composition in predicting future lipid patterns and cardiovascular disease risk.

An association of CSF apolipoprotein E glycosylation and amyloid-beta 42 in individuals who carry the APOE4 allele.

Carrying the apolipoprotein E (ApoE) Ɛ4 allele is associated with an increased risk of cerebral amyloidosis and late-onset Alzheimer's disease, but the degree to which apoE glycosylation affects its development is not clear. In a previous pilot study, we identified distinct total and secondary isoform-specific cerebral spinal fluid (CSF) apoE glycosylation profiles, with the E4 isoform having the lowest glycosylation percentage (E2 > E3 > E4). In this work, we extend the analysis to a larger cohort of individuals (n = 106), utilizing matched plasma and CSF samples with clinical measures of AD biomarkers. The results confirm the isoform-specific glycosylation of apoE in CSF, resulting from secondary CSF apoE glycosylation patterns. CSF apoE glycosylation percentages positively correlated with CSF Aß42 levels (r = 0.53, p < 0.0001). These correlations were not observed for plasma apoE glycosylation. CSF total and secondary apoE glycosylation percentages also correlated with the concentration of CSF small high-density lipoprotein particles (s-HDL-P), which we have previously shown to be correlated with CSF Aß42 levels and measures of cognitive function. Desialylation of apoE purified from CSF showed reduced Aß42 degradation in microglia with E4 > E3 and increased binding affinity to heparin. These results indicate that apoE glycosylation has a new and important role in influencing brain Aß metabolism and can be a potential target of treatment.

Plasma proteoforms of apolipoproteins C-I and C-II are associated with plasma lipids in the Multi-Ethnic Study of Atherosclerosis.

Apolipoproteins (apo) C-I and C-II are key regulators of triglyceride and HDL metabolism. Both exist as full-size native and truncated (apoC-I'; apoC-II') posttranslational proteoforms. However, the determinants and the role of these proteoforms in lipid metabolism are unknown. Here, we measured apoC-I and apoC-II proteoforms by mass spectrometry immunoassay in baseline and 10-year follow-up plasma samples from the Multi-Ethnic Study of Atherosclerosis. We found that baseline total apoC-I (mean = 9.2 mg/dl) was lower in African Americans (AA), Chinese Americans (CA), and Hispanics (by 1.8; 1.0; 1.0 mg/dl vs. whites), higher in women (by 1.2 mg/dl), and positively associated with plasma triglycerides and HDL. Furthermore, we observed that the truncated-to-native apoC-I ratio (apoC-I'/C-I) was lower in CA, negatively associated with triglycerides, and positively associated with HDL. We determined that total apoC-II (8.8 mg/dl) was lower in AA (by 0.8 mg/dl) and higher in CA and Hispanics (by 0.5 and 0.4 mg/dl), positively associated with triglycerides, and negatively associated with HDL. In addition, apoC-II'/C-II was higher in AA and women, negatively associated with triglycerides, and positively associated with HDL. We showed that the change in triglycerides was positively associated with changes in total apoC-I and apoC-II and negatively associated with changes in apoC-I'/C-I and apoC-II'/C-II, whereas the change in HDL was positively associated with changes in total apoC-I and apoC-II'/C-II and negatively associated with change in total apoC-II. This study documents racial/ethnic variation in apoC-I and apoC-II plasma levels and highlights apolipoprotein posttranslational modification as a potential regulator of plasma lipids.

A novel substitution of proline (P32L) destabilises ß2-microglobulin inducing hereditary systemic amyloidosis.

BACKGROUND: ß2-microglobulin amyloidosis was first described in the 1980s as a protein deposition disease associated with long-term haemodialysis. More recently, two inherited forms resulting from separate point mutations in the ß2-microglobulin gene have been identified. In this report, we detail a novel ß2M variant, P32L, caused by a unique dinucleotide mutation that is linked to systemic hereditary ß2-microglobulin amyloidosis. METHODS: Three family members from a Portuguese kinship featured cardiomyopathy, requiring organ transplantation in one case, along with soft tissue involvement; other involvements included gastrointestinal, neuropathic and sicca syndrome. In vitro studies with recombinant P32L, P32G, D76N and wild-type ß2-microglobulin were undertaken to compare the biophysical properties of the proteins. RESULTS: The P32L variant was caused by the unique heterozygous dinucleotide mutation c.154_155delinsTT. Amyloid disease featured lowered serum ß2-microglobulin levels with near equal amounts of circulating P32L and wild-type proteins; amyloid deposits were composed exclusively of P32L variant protein. In vitro studies of P32L demonstrated thermodynamic and chemical instability and enhanced susceptibility to proteolysis with rapid formation of pre-fibrillar oligomeric structures by N- and C-terminally truncated species under physiological conditions. CONCLUSIONS: This work provides both clinical and experimental evidence supporting the critical role of P32 residue replacement in ß2M amyloid fibrillogenesis.

Distinct patterns of apolipoprotein C-I, C-II, and C-III isoforms are associated with markers of Alzheimer's disease.

Apolipoproteins C-I, C-II, and C-III interact with ApoE to regulate lipoprotein metabolism and contribute to Alzheimer's disease pathophysiology. In plasma, apoC-I and C-II exist as truncated isoforms, while apoC-III exhibits multiple glycoforms. This study aimed to 1) delineate apoC-I, C-II, and C-III isoform profiles in cerebrospinal fluid (CSF) and plasma in a cohort of nondemented older individuals (n = 61), and 2) examine the effect of APOE4 on these isoforms and their correlation with CSF Aß42, a surrogate of brain amyloid accumulation. The isoforms of the apoCs were immunoaffinity enriched and measured with MALDI-TOF mass spectrometry, revealing a significantly higher percentage of truncated apoC-I and apoC-II in CSF compared with matched plasma, with positive correlation between CSF and plasma. A greater percentage of monosialylated and disialylated apoC-III isoforms was detected in CSF, accompanied by a lower percentage of the two nonsialylated apoC-III isoforms, with significant linear correlations between CSF and plasma. Furthermore, a greater percentage of truncated apoC-I in CSF and apoC-II in plasma and CSF was observed in individuals carrying at least one APOE Ɛ4 allele. Increased apoC-I and apoC-II truncations were associated with lower CSF Aß42. Finally, monosialylated apoC-III was lower, and disialylated apoC-III greater in the CSF of Ɛ4 carriers. Together, these results reveal distinct patterns of the apoCs isoforms in CSF, implying CSF-specific apoCs processing. These patterns were accentuated in APOE Ɛ4 allele carriers, suggesting an association between APOE4 genotype and Alzheimer's disease pathology with apoCs processing and function in the brain.

Simple and Fast Assay for Apolipoprotein E Phenotyping and Glycotyping: Discovering Isoform-Specific Glycosylation in Plasma and Cerebrospinal Fluid.

BACKGROUND: The mechanisms of how APOEɛ4 allele (APOE4) increases the risk of Alzheimer's disease (AD) pathology have not been fully elucidated. In cerebrospinal fluid (CSF), apoE is heavily glycosylated. OBJECTIVE: To determine the impact of APOE genotype on the relative abundance of apoE protein isoforms and their specific glycosylation patterns in CSF and plasma via a newly developed mass spectrometric immunoassay (MSIA) assay. METHODS: Total glycosylation and isoform-specific glycosylation were analyzed in plasma and CSF from a group of non-demented older individuals (n = 22), consisting of homozygous ɛ3 and ɛ4 or heterozygous ɛ3/ɛ4, ɛ2/ɛ3, or ɛ2/ɛ4 carriers. The glycan structures were further confirmed after treatment with sialidase. RESULTS: In heterozygous individuals, the apoE3/E2, E4/E2, and E4/E3 isoform ratios were all significantly lower in plasma compared to CSF. For all individuals, a single O-linked glycan was observed in plasma, while two glycans (of the same type) per apoE were observed in CSF. The ratio of glycosylated to total apoE was greater in CSF compared to plasma for all apoE isoforms. In plasma and CSF, a trend of decreasing glycosylation was observed from apoE2 > apoE3 > apoE4. The difference in the percentage of secondary glycosylation in CSF was significantly greater in apoE4 compared to the other isoforms. CONCLUSION: The new MSIA apoE assay robustly distinguishes among apoE isoforms and glycoforms in plasma and CSF. ApoE4 is the predominant isoform and least glycosylated in CSF. Assessing apoE isoform-specific glycosylation by MSIA may help clarify brain apoE metabolism and AD risk.

Complexity, cost, and content - three important factors for translation of clinical protein mass spectrometry tests, and the case for apolipoprotein C-III proteoform testing.

Complexity, cost, and content are three important factors that can impede translation of clinical protein mass spectrometry (MS) tests at a larger scale. Complexity stems from the many components/steps involved in bottom-up protein MS workflows, making them significantly more complicated than enzymatic immunoassays (EIA) that currently dominate clinical testing. This complexity inevitably leads to increased costs, which is detrimental in the price-competitive clinical marketplace. To successfully compete, new clinical protein MS tests need to offer something new and unique that EIAs cannot - a new content of proteoform detection. The preferred method for proteoform profiling is intact protein MS analysis, in which all proteins are measured as intact species thus allowing discovery of new proteoforms. To illustrate the importance of intact proteoform testing with MS and its potential clinical implications, we discuss here recent findings from multiple studies on the distribution of apolipoprotein C-III proteoforms and their correlations with key clinical measures of dyslipidemia. Such studies are only made possible with assays that are low in cost, avoid unnecessary complexity, and are unique in providing the content of proteoforms.

ApoC-III Glycoforms Are Differentially Cleared by Hepatic TRL (Triglyceride-Rich Lipoprotein) Receptors.

OBJECTIVE: ApoC-III (apolipoprotein C-III) glycosylation can predict cardiovascular disease risk. Higher abundance of disialylated (apoC-III2) over monosialylated (apoC-III1) glycoforms is associated with lower plasma triglyceride levels. Yet, it remains unclear whether apoC-III glycosylation impacts TRL (triglyceride-rich lipoprotein) clearance and whether apoC-III antisense therapy (volanesorsen) affects distribution of apoC-III glycoforms. Approach and Results: To measure the abundance of human apoC-III glycoforms in plasma over time, human TRLs were injected into wild-type mice and mice lacking hepatic TRL clearance receptors, namely HSPGs (heparan sulfate proteoglycans) or both LDLR (low-density lipoprotein receptor) and LRP1 (LDLR-related protein 1). ApoC-III was more rapidly cleared in the absence of HSPG (t1/2=25.4 minutes) than in wild-type animals (t1/2=55.1 minutes). In contrast, deficiency of LDLR and LRP1 (t1/2=56.1 minutes) did not affect clearance of apoC-III. After injection, a significant increase in the relative abundance of apoC-III2 was observed in HSPG-deficient mice, whereas the opposite was observed in mice lacking LDLR and LRP1. In patients, abundance of plasma apoC-III glycoforms was assessed after placebo or volanesorsen administration. Volanesorsen treatment correlated with a statistically significant 1.4-fold increase in the relative abundance of apoC-III2 and a 15% decrease in that of apoC-III1. The decrease in relative apoC-III1 abundance was strongly correlated with decreased plasma triglyceride levels in patients. CONCLUSIONS: Our results indicate that HSPGs preferentially clear apoC-III2. In contrast, apoC-III1 is more effectively cleared by LDLR/LRP1. Clinically, the increase in the apoC-III2/apoC-III1 ratio on antisense lowering of apoC-III might reflect faster clearance of apoC-III1 because this metabolic shift associates with improved triglyceride levels.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Top-down mass spectrometric immunoassay for human insulin and its therapeutic analogs.

Measurement of insulin and its therapeutic analogs is important in diabetes, hypoglycemia, sports anti-doping and toxicology. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogs. Because of their unique sequences and masses, these analogs are readily measured and distinguished with mass spectrometric (MS) assays. Reviewed here is an insulin mass spectrometric immunoassay (MSIA) that combines micro-scale immunoaffinity capture with sensitive MS detection of insulin and its therapeutic analogs. An antibody reactive to all insulin analogs was used to affinity capture the insulin analogs. Following elution, insulins were detected with MALDI-TOF MS or LC-MS analysis. Isotopic resolution for insulin was achieved for both MS techniques, and several insulin analogs were detected at unique m/z signals. Porcine insulin, spiked in all samples, served as an internal reference standard for quantification. Linear standard curves spanning three orders of magnitude were obtained, with limits of detection of 15pM for the MALDI-TOF MS and 1.5pM for the LC-MS. This insulin assay was capable of detecting and quantifying not only human endogenous insulin, but also most of the therapeutic insulin analogs, which could find use in diagnosis of severe hypoglycemia and in sports anti-doping. SIGNIFICANCE: Insulin replacement therapy consists of injection of long- or fast-acting insulin analogs with slightly modified primary sequences compared to human insulin. Assays that are capable of detecting all insulin analogs are desired, not only for medical management of diabetes and severe hypoglycemia but also for sports anti-doping and toxicology.

Mass Spectrometric Studies of Apolipoprotein Proteoforms and Their Role in Lipid Metabolism and Type 2 Diabetes.

Apolipoproteins function as structural components of lipoprotein particles, cofactors for enzymes, and ligands for cell-surface receptors. Most of the apoliporoteins exhibit proteoforms, arising from single nucleotide polymorphisms (SNPs) and post-translational modifications such as glycosylation, oxidation, and sequence truncations. Reviewed here are recent studies correlating apolipoproteins proteoforms with the specific clinical measures of lipid metabolism and cardiometabolic risk. Targeted mass spectrometric immunoassays toward apolipoproteins A-I, A-II, and C-III were applied on large cross-sectional and longitudinal clinical cohorts. Several correlations were observed, including greater apolipoprotein A-I and A-II oxidation in patients with diabetes and cardiovascular disease, and a divergent apoC-III proteoforms association with plasma triglycerides, indicating significant differences in the metabolism of the individual apoC-III proteoforms. These are the first studies of their kind, correlating specific proteoforms with clinical measures in order to determine their utility as potential clinical biomarkers for disease diagnosis, risk stratification, and therapy decisions. Such studies provide the impetus for the further development and clinical translation of MS-based protein tests.

Human proteoforms as new targets for clinical mass spectrometry protein tests.

INTRODUCTION: Only about dozen mass spectrometry (MS) protein tests have been translated into clinical laboratories since the MALDI and ESI approaches were developed thirty years ago. While the cost and complexity of these assays are important factors impeding their clinical adoption, new content generated via proteoforms detection could provide the impetus for further development and translation. Areas covered: Provided here are several examples of MS-based protein assays capable of detecting proteoforms, including those for B-type natriuretic peptide (BNP) and parathyroid hormone (PTH). The evidence suggests that the ability to detect proteoforms is not enough to drive the clinical adoption of the MS-based tests - clinical utility of those proteoforms needs to be demonstrated first. Along those lines, recent efforts to discover, clinically validate, and initiate translation of novel proteoform biomarkers such as those of apolipoprotein C-III will be discussed. Expert commentary: MS protein tests face a challenging future. Both the sample preparation steps and the MS platforms need to be simplified to bring the cost per test down, and then the new content brought by the detection of proteoforms will drive the proliferation of these MS tests - first in clinical utility studies and then for routine diagnostics.

Changes in low-density lipoprotein size phenotypes associate with changes in apolipoprotein C-III glycoforms after dietary interventions.

BACKGROUND: The presence of small dense low-density lipoprotein (LDL) is associated with obesity, type II diabetes, and an increased risk for cardiovascular disease. Apolipoprotein C-III (apoC-III) is involved in the formation of small dense LDL, but the exact mechanisms are still not well defined. ApoC-III is a glycosylated apolipoprotein, with 3 major glycoforms: apoC-III0, apoC-III1, and apoC-III2 that contain 0, 1, or 2 molecules of sialic acid, respectively. In our previous work, we reported an association among apoC-III0 and apoC-III1, but not apoC-III2 with fasting plasma triglyceride levels in obesity and type II diabetes. OBJECTIVE: The goal of this study was to determine the relationship between changes in the major apoC-III glycoforms and small dense LDL levels after dietary interventions. METHODS: Mass spectrometric immunoassay was performed on fasting plasma samples from 61 subjects who underwent either a high-carbohydrate diet (n = 34) or a weight loss intervention (n = 27). RESULTS: After both dietary interventions, changes in total apoC-III concentrations were associated with changes in LDL peak particle diameter (r = -0.58, P < .0001). Increases in total apoC-III concentrations after the high-carbohydrate diet were associated with decreases in LDL size (r = -0.53, P = .001), and decreases in apoC-III concentrations after weight loss were associated with increases in LDL peak particle diameter (r = -0.54, P = .004). Changes in concentrations of apoC-III1 and apoC-III0, but not apoC-III2, were associated with changes in LDL peak particle diameter in both the weight loss and high-carbohydrate interventions. CONCLUSIONS: We conclude that apoC-III0 and apoC-III1, but not apoC-III2 are associated with the formation of small dense LDL.

Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring.

Tuberculosis (TB) is a major global health threat, resulting in an urgent unmet need for a rapid, non-sputum-based quantitative test to detect active Mycobacterium tuberculosis (Mtb) infections in clinically diverse populations and quickly assess Mtb treatment responses for emerging drug-resistant strains. We have identified Mtb-specific peptide fragments and developed a method to rapidly quantify their serum concentrations, using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) and high-throughput mass spectrometry (MS) to enhance sensitivity and specificity. NanoDisk-MS diagnosed active Mtb cases with high sensitivity and specificity in a case-control study with cohorts reflecting the complexity of clinical practice. Similar robust sensitivities were obtained for cases of culture-positive pulmonary TB (PTB; 91.3%) and extrapulmonary TB (EPTB; 92.3%), and the sensitivities obtained for culture-negative PTB (82.4%) and EPTB (75.0%) in HIV-positive patients significantly outperformed those reported for other available assays. NanoDisk-MS also exhibited high specificity (87.1-100%) in both healthy and high-risk groups. Absolute quantification of serum Mtb antigen concentration was informative in assessing responses to antimycobacterial treatment. Thus, a NanoDisk-MS assay approach could significantly improve the diagnosis and management of active TB cases, and perhaps other infectious diseases as well.

Mass spectrometry protein tests: ready for prime time (or not).

INTRODUCTION: Mass spectrometry has played an important role in protein biomarker discovery. Yet, very few of the candidate biomarkers have been validated, and mass spectrometry-based protein tests have not made a significant inroad into clinical laboratories. Areas covered: Offered here is a unique perspective on the future of mass spectrometry protein tests, in view of the following determinants: the true demand for such clinical tests, end-users requirements, platforms and systems design, sample preparation bottlenecks, analytical and clinical validation, and regulatory approval. Expert commentary: Fresh thoughts and attitudes toward MS protein tests are required in order to move them toward clinical utilization and diagnostic use en masse, with critical emphasis on content, simplicity and cost. In its current format and state of the art, they are simply not ready for prime time.

Posttranslational modifications of apolipoprotein A-II proteoforms in type 2 diabetes.

BACKGROUND: Apolipoprotein A-II (apoA-II) is the second most abundant protein in high-density lipoprotein particles. However, it exists in plasma in multiple forms. The effect of diabetes on apoA-II proteoforms is not known. OBJECTIVE: Our objective was to characterize plasma apoA-II proteoforms in participants with and without type 2 diabetes. METHODS: Using a novel mass spectrometric immunoassay, the relative abundance of apoA-II proteoforms was examined in plasma of 30 participants with type 2 diabetes and 25 participants without diabetes. RESULTS: Six apoA-II proteoforms (monomer, truncated TQ monomer, truncated Q monomer, dimer, truncated Q dimer, and truncated 2Qs dimer) and their oxidized proteoforms were identified. The ratios of oxidized monomer and all oxidized proteoforms to the native apoA-II were significantly greater in the diabetic group (P = .004 and P = .005, respectively) compared with the nondiabetic group. CONCLUSION: The relative abundance of oxidized apoA-II is significantly increased in type 2 diabetes.

Mass spectrometric immunoassays for discovery, screening and quantification of clinically relevant proteoforms.

Human proteins can exist as multiple proteoforms with potential diagnostic or prognostic significance. MS top-down approaches are ideally suited for proteoforms identification because there is no prerequisite for a priori knowledge of the specific proteoform. One such top-down approach, termed mass spectrometric immunoassay utilizes antibody-derivatized microcolumns for rapid and contained proteoforms isolation and detection via MALDI-TOF MS. The mass spectrometric immunoassay can also provide quantitative measurement of the proteoforms through inclusion of an internal reference standard into the analytical sample, serving as normalizer for all sample processing and data acquisition steps. Reviewed here are recent developments and results from the application of mass spectrometric immunoassays for discovery of clinical correlations of specific proteoforms for the protein biomarkers RANTES, retinol binding protein, serum amyloid A and apolipoprotein C-III.

Development of quantitative mass spectrometric immunoassay for serum amyloid A.

OBJECTIVE: Proteins can exist as multiple proteoforms in vivo that can have important roles in physiological and pathological states. METHODS: We present the development and characterization of mass spectrometric immunoassay (MSIA) for quantitative determination of serum amyloid A (SAA) proteoforms. RESULTS: Intra- and inter-day precision revealed CVs <10%. Against existing SAA ELISA, the developed MSIA showed good correlation according to the Altman-Bland plot. Individual concentrations of the SAA proteoforms across a cohort of 170 samples revealed 7 diverse SAA polymorphic types and 12 different proteoforms. CONCLUSION: The new SAA MSIA enables parallel analysis of SAA polymorphisms and quantification of all expressed SAA proteoforms, in a high-throughput and time-efficient manner.