Interlaboratory Comparison of Antibody-Free LC-MS/MS Measurements of C-peptide and Insulin.

BACKGROUND: The enhanced precision and selectivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it an attractive alternative to certain clinical immunoassays. Easily transferrable work flows could help facilitate harmonization and ensure high-quality patient care. We aimed to evaluate the interlaboratory comparability of antibody-free multiplexed insulin and C-peptide LC-MS/MS measurements. METHODS: The laboratories that comprise the Targeted Mass Spectrometry Assays for Diabetes and Obesity Research (TaMADOR) consortium verified the performance of a validated peptide-based assay (reproducibility, linearity, and lower limit of the measuring interval [LLMI]). An interlaboratory comparison study was then performed using shared calibrators, de-identified leftover laboratory samples, and reference materials. RESULTS: During verification, the measurements were precise (2.7% to 3.7%CV), linear (4 to 15 ng/mL for C-peptide and 2 to 14 ng/mL for insulin), and sensitive (LLMI of 0.04 to 0.10 ng/mL for C-peptide and 0.03 ng/mL for insulin). Median imprecision across the 3 laboratories was 13.4% (inter-quartile range [IQR] 11.6%) for C-peptide and 22.2% (IQR 20.9%) for insulin using individual measurements, and 10.8% (IQR 8.7%) and 15.3% (IQR 14.9%) for C-peptide and insulin, respectively, when replicate measurements were averaged. Method comparison with the University of Missouri reference method for C-peptide demonstrated a robust linear correlation with a slope of 1.044 and r2 = 0.99. CONCLUSIONS: Our results suggest that combined LC-MS/MS measurements of C-peptide and insulin are robust and adaptable and that standardization with a reference measurement procedure could allow accurate and precise measurements across sites, which could be important to diabetes research and help patient care in the future.

Comprehensive profiling and kinetic studies of glycated lysine residues in human serum albumin.

Lysine residues of proteins slowly react with glucose forming Amadori products. In hyperglycemic conditions, such as diabetes mellitus, this non-enzymatic glycation becomes more pervasive causing severe medical complications. The structure and conformation of a protein predisposes lysine sites to differing reactivity influenced by their steric availability and amino acid microenvironment. The goal of our study was to identify these sites in albumin and measure glycation affinities of lysine residues. We applied a bottom-up approach utilizing a combination of three LC-MS instruments: timsTOF, Orbitrap, and QTRAP. To prove applicability to samples of varying glycemic status, we compared in vitro glycated and non-glycated HSA, as well as diabetic and non-diabetic individual samples. The analysis of lysine glycation affinities based on peptide intensities provide a semi-quantitative approach, as the results depend on the mass spectrometry platform used. We found that glycation levels based on multiple reaction monitoring (MRM) quantitation better reflect individual glycemic status and that the glycation percentage for each site is in linear relation to all other sites. To develop an approach which more accurately reflects glycation affinity, we developed a kinetics model which uses results from stable isotope dilution HPLC-MRM methodology. Through glycation of albumin at different glucose concentrations, we determine the rate constants of glycation for every lysine residue by simultaneous comparative analysis.

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed.

Novel synthetic approach to charge-compensated phosphonio-nido-carboranes. Synthesis and structural characterization of neutral mono and bis(phosphonio) nido-ortho-carboranes.

A number of monosubstituted n-(triphenylphosphonio)-7,8-dicarba-nido-undecaboranes (2a, n = 1; 2b, n = 3; 2c, n = 5; 2d, n = 9) were prepared via a cross-coupling reaction between the tetrabutylammonium iodo-7,8-dicarba-nido-undecaborates (1a-d) and PPh3 in the presence of a Pd(PPh3)4 catalyst. The substitution rate was found to depend on the iodine position in the carborane cage. Under similar conditions, the reaction of 5,6-diiodo- (3) and 9,11-diiodo-7,8-dicarba-nido-undecaborate (5) anions exclusively yielded the monosubstitution products 5-iodo-6-(triphenylphosphonio)-7,8-dicarba-nido-undecaborane (4) and 9-iodo-11-(triphenylphosphonio)-7,8-dicarba-nido-undecaborane (6), respectively. The reaction of tetrabutylammonium 6,9-diiodo-7,8-dicarba-nido-undecaborate (7) exclusively produced the phosphine substitution product in the open face of the nido-carborane, 6-iodo-9-triphenylphosphonio-7,8-dicarba-nido-undecaborane (8). The addition of a base (Cs2CO3, NaH) to the reactions of 3 and 5 with PPh3 afforded the corresponding bis(triphenylphosphonio)-7,8-dicarba-nido-undecaboranes, 9 and 10. Compound 10 was also prepared from 6 using the general procedure. The reaction of the triiodocarborane tetrabutylammonium 5,6,9-triiodo-7,8-dicarba-nido-undecaborate (11) with excess PPh3 in the presence of Cs2CO3 and Pd(PPh3)4 only produced neutral 5-iodo-6,9-bis(triphenylphosphonio)-7,8-dicarba-nido-undecaborane (12); no positively charged tris(phosphonio) species formed. The compositions of all prepared compounds were determined by multinuclear NMR spectroscopy and high-resolution mass spectrometry. The structures of compounds 2c, 6, 8, 9, and 12 were established by the X-ray diffraction analysis of single crystals.

Diversification of EPR signatures in Site Directed Spin Labeling using a ß-phosphorylated nitroxide.

Site Directed Spin Labeling (SDSL) combined with EPR spectroscopy is a very powerful approach to investigate structural transitions in proteins in particular flexible or even disordered ones. Conventional spin labels are based on nitroxide derivatives leading to classical 3-line spectra whose spectral shapes are indicative of the environment of the labels and thus constitute good reporters of structural modifications. However, the similarity of these spectral shapes precludes probing two regions of a protein or two partner proteins simultaneously. To overcome the limitation due to the weak diversity of nitroxide label EPR spectral shapes, we designed a new spin label based on a ß-phosphorylated nitroxide giving 6-line spectra. This paper describes the synthesis of this new spin label, its grafting at four different positions of a model disordered protein able to undergo an induced α-helical folding and its characterization by EPR spectroscopy. For comparative purposes, a classical nitroxide has been grafted at the same positions of the model protein. The ability of the new label to report on structural transitions was evaluated by analyzing the spectral shape modifications induced either by the presence of a secondary structure stabilizer (trifluoroethanol) or by the presence of a partner protein. Taken together the results demonstrate that the new phosphorylated label gives a very distinguishable signature which is able to report from subtle to larger structural transitions, as efficiently as the classical spin label. As a complementary approach, molecular dynamics (MD) calculations were performed to gain further insights into the binding process between the labeled NTAIL and PXD. MD calculations revealed that the new label does not disturb the interaction between the two partner proteins and reinforced the conclusion on its ability to probe different local environments in a protein. Taken together this study represents an important step forward in the extension of the panoply of SDSL-EPR approaches.

Physio-chemical Modifications to Re-engineer Small Extracellular Vesicles for Targeted Anticancer Therapeutics Delivery and Imaging.

Cancer theranostics developed through nanoengineering applications are essential for targeted oncologic interventions in the new era of personalized and precision medicine. Recently, small extracellular vesicles (sEVs) have emerged as an attractive nanoengineering platform for tumor-directed anticancer therapeutic delivery and imaging of malignant tumors. These natural nanoparticles have multiple advantages over synthetic nanoparticle-based delivery systems, such as intrinsic targeting ability, less immunogenicity, and a prolonged circulation time. Since the inception of sEVs as a viable replacement for liposomes (synthetic nanoparticles) as a drug delivery vehicle, many studies have attempted to further the therapeutic efficacy of sEVs. This article discusses engineering strategies for sEVs using physical and chemical methods to enhance their anticancer therapeutic delivery performance. We review physio-chemical techniques of effective therapeutic loading into sEV, sEV surface engineering for targeted entry of therapeutics, and its cancer environment sensitive release inside the cells/organ. Next, we also discuss the novel hybrid sEV systems developed by a combination of sEVs with lipid and metal nanoparticles to garner each component's benefits while overcoming their drawbacks. The article extensively analyzes multiple sEV labeling techniques developed and investigated for live tracking or imaging sEVs. Finally, we discuss the theranostic potential of engineered sEVs in future cancer care regimens.

Protein glycation in diabetes mellitus.

Diabetes mellitus is the ninth leading cause of mortality worldwide. It is a complex disease that manifests as chronic hyperglycemia. Glucose exposure causes biochemical changes at the proteome level as reflected in accumulation of glycated proteins. A prominent example is hemoglobin A1c (HbA1c), a glycated protein widely accepted as a diabetic indicator. Another emerging biomarker is glycated albumin which has demonstrated utility in situations where HbA1c cannot be used. Other proteins undergo glycation as well thus impacting cellular function, transport and immune response. Accordingly, these glycated counterparts may serve as predictors for diabetic complications and thus warrant further inquiry. Fortunately, modern proteomics has provided unique analytic capability to enable improved and more comprehensive exploration of glycating agents and glycated proteins. This review broadly covers topics from epidemiology of diabetes to modern analytical tools such as mass spectrometry to facilitate a better understanding of diabetes pathophysiology. This serves as an attempt to connect clinically relevant questions with findings of recent proteomic studies to suggest future avenues of diabetes research.

Higher degree of glycation of hemoglobin S compared to hemoglobin A measured by mass spectrometry: Potential impact on HbA1c testing.

BACKGROUND: Glycated hemoglobin (GHb), reported as HbA1c, is used as marker of long-term glycemia for diabetic patients. HbA1c results from boronate affinity methods are generally considered to be unaffected by most hemoglobin variants; this assumes comparable glycation of variant and non-variant (HbAA) hemoglobins. In this report, glycation of HbA beta chain (ßA) and HbS beta chain (ßS) for the most common Hb variant trait (HbAS) are examined. METHODS: We analyzed 41 blood samples from subjects with HbAS, both with and without diabetes. Using LC-MS, ratios of glycated HbS to glycated HbA were determined by comparison of areas under the curves from extracted ion chromatograms. RESULTS: Glycation of ßS chains was significantly higher (p<0.001) than ßA chains; this difference was consistent across subjects. Total (α+ß) glycated HbAS was theoretically estimated to be ~5% higher than glycated HbAA. CONCLUSION: This novel mass-spectrometric approach described allows for relative quantification of glycated forms of ßS and ßA. Although ßS glycation was significantly higher than that of ßA, the difference in total glycation of HbAS versus HbAA was smaller and unlikely to impact clinical interpretation of boronate affinity HbA1c results. These data support the continued use of boronate affinity to measure HbA1c in patients with HbAS.

Carborane-derived local anesthetics are isomer dependent.

Clinically there is a need for local anesthetics with a greater specificity of action on target cells and longer duration. We have synthesized a series of local anesthetic derivatives we call boronicaines in which the aromatic phenyl ring of lidocaine was replaced with ortho-, meta-, C,C'-dimethyl meta- and para-carborane clusters. The boronicaine derivatives were tested for their analgesic activity and compared with lidocaine using standard procedures in mice following a plantar injection. The compounds differed in their analgesic activity in the following order: ortho-carborane = C,C'-dimethyl meta-carborane > para-carborane > lidocaine > meta-carborane derivative. Both ortho-boronicaine and C,C'-dimethyl meta-boronicaine had longer durations of analgesia than lidocaine. Differences in analgesic efficacies are rationalized by variations in chemical structure and protein binding characteristics.

Novel iodinated carboranes: synthesis of the 8-iodo-7,9-dicarba-nido-undecaborate anion and 2-iodo-1,7-dicarba-closo-dodecaborane.

Electrophilic iodination of the 7,9-dicarba-nido-undecaborate anion with molecular iodine in the presence of AlCl3 generated a new carborane anion-8-iodo-7,9-dicarba-nido-undecaborate-in excellent yield. The capping of the new anion with HBCl2 yielded a previously unknown neutral iodinated carborane, 2-iodo-1,7-dicarba-closo-dodecaborane.