Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry.

Myelin basic protein (MBP) plays an important structural and functional role in the neuronal myelin sheath. Translated MBP exhibits extreme microheterogeneity with numerous alternative splice variants (ASVs) and post-translational modifications (PTMs) reportedly tied to central nervous system maturation, myelin stability, and the pathobiology of various de- and dys-myelinating disorders. Conventional bioanalytical tools cannot efficiently examine ASV and PTM events simultaneously, which limits understanding of the role of MBP microheterogeneity in human physiology and disease.

Proteoform analysis of lipocalin-type prostaglandin D-synthase from human cerebrospinal fluid by isoelectric focusing and superficially porous liquid chromatography with Fourier transform mass spectrometry.

Lipocalin-type prostaglandin D-synthase (L-PGDS) in cerebrospinal fluid contributes to the maturation and maintenance of the CNS. L-PGDS PTMs may contribute to pathobiology of different CNS diseases, but methods to monitor its proteoforms are limited. Herein, we combined off-gel IEF and superficially porous LC (SPLC) with Fourier transform MS to characterize common cerebrospinal fluid L-PGDS proteoforms.

Measurement of blood protease kinetic parameters with self-assembled monolayer ligand binding assays and label-free MALDI-TOF MS.

We report novel ligand binding assay (LBA) surface modalities that permit plasma protease catalytic efficiency (kcat/km) determination by MALDI-TOF MS without the use of liquid chromatography or internal standards such as chemical or metalized labels. Two model LBAs were constructed on planar self-assembled monolayers (SAMs) and used to evaluate the clinically relevant metalloprotease ADAMTS-13 kinetics in plasma. The SAM chemistries were designed to improve biosampling efficiency by minimization of nonspecific adsorption of abundant proteins present at ~100,000× the concentration of the endogenous enzyme.

Top-down mass spectrometry on tissue extracts and biofluids with isoelectric focusing and superficially porous silica liquid chromatography.

Top-down mass spectrometry (MS) has emerged as a powerful complement to peptide-based proteomics. Despite advancements, the field has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations, we developed a two-dimensional (2D) chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient and superficially porous liquid chromatography (SPLC).

Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ.

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells.

Surface preparation strategies for improved parallelization and reproducible MALDI-TOF MS ligand binding assays.

Immunoassays are employed in academia and the healthcare and biotech industries for high-throughput, quantitative screens of biomolecules. We have developed monolayer-based immunoassays for MALDI-TOF MS. To improve parallelization, we adapted the workflow to photolithography-generated arrays.

MS-based ligand binding assays with speed, sensitivity, and specificity.

Immunoassays are widely used in biochemical/clinical laboratories owing to their simplicity, speed, and sensitivity. We combined self-assembled monolayer-based immunoassays with MALDI-TOF MS to show that high-fidelity surface preparations with a novel matrix deposition/crystallization technique permits quantitative analysis of monolayer-bound antigens at picomolar detection limits. Calibration curves for intact proteins are possible over a broad concentration range and improved specificity of MS-immunoassays is highlighted by simultaneous label-free quantitation of ligand-bound protein complexes.

Thin-layer matrix sublimation with vapor-sorption induced co-crystallization for sensitive and reproducible SAMDI-TOF MS analysis of protein biosensors.

Coupling immunoassays on self-assembled monolayers (SAMs) to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) provides improved assay selectivity compared with traditional photometric detection techniques. We show that thin-layer-transfer (TLT) of α-cyano-4-hydroxycinnaminic acid (CHCA) MALDI matrix via vacuum sublimation followed by organic solvent-based vapor-sorption induced co-crystallization (VIC) results in unique matrix/analyte co-crystallization tendencies that optimizes assay reproducibility and sensitivity. Unique matrix crystal morphologies resulted from VIC solvent vapors, indicating nucleation and crystal growth characteristics depend upon VIC parameters.

Low placental growth factor across pregnancy identifies a subset of women with preterm preeclampsia: type 1 versus type 2 preeclampsia?

Preeclampsia is a heterogeneous syndrome affecting 3% to 5% of all pregnancies. An imbalance of the antiangiogenic and proangiogenic factors, soluble receptor fms-like tyrosine kinase 1 and placental growth factor (PGF), is thought to contribute to the pathophysiology of preeclampsia. Maternal plasma PGF and soluble receptor fms-like tyrosine kinase 1 were quantified by specific immunoassays in cross-sectional samples from 130 preeclamptic subjects and 342 normotensive controls at delivery and longitudinally in samples from 50 women who developed preeclampsia and 250 normotensive controls.

Sensitive and reproducible intact mass analysis of complex protein mixtures with superficially porous capillary reversed-phase liquid chromatography mass spectrometry.

The compatibility of superficially porous (SP) resin for label-free intact protein analysis with online capillary LC/MS is demonstrated to give improved chromatographic resolution, sensitivity, and reproducibility. The robustness of the platform was measured against several samples of varying complexity and sample loading amount. The results indicate that capillary SP columns provide high loading capacities and that ∼6 s chromatographic peak widths are typical for standard proteins in simple mixtures and proteins isolated from cell and tissue lysates.

Maternal endothelial progenitor colony-forming units with macrophage characteristics are reduced in preeclampsia.

Background: Endothelial progenitor cells (EPCs) provide paracrine support to the vascular endothelium and may also replace damaged or senescent endothelial cells. Low numbers of endothelial progenitor colony-forming units (CFU-ECs) in culture are a predictive biomarker of vascular disease. We hypothesized that the number of CFU-ECs derived from maternal blood are decreased in women with preeclampsia compared to normal pregnancy.