Interlaboratory reproducibility of selective reaction monitoring assays using multiple upfront analyte enrichment strategies.

Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays.

Discrimination of ischemic and hemorrhagic strokes using a multiplexed, mass spectrometry-based assay for serum apolipoproteins coupled to multi-marker ROC algorithm.

Purpose: Typically, apolipoproteins are individually measured in blood by immunoassay. In this report, we describe the development of a multiplexed selected reaction monitoring (SRM) based assay for a panel of apolipoproteins and its application to a clinical cohort of samples derived from acute stroke patients.

Experimental Design: An SRM assay for a panel of nine apolipoproteins was developed on a triple quadrupole mass spectrometer.

On-resin N-methylation of cyclic peptides for discovery of orally bioavailable scaffolds.

Backbone N-methylation is common among peptide natural products and has a substantial impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability.

A large network of interconnected signaling pathways in human ovarian follicles is supported by the gene expression activity of the granulosa cells.

Human follicular fluid (hFF), as an extra oocyte microenvironment, is essential to the biological processes of oocyte development. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 426 proteins as consistently present in hFF from different participants. According to our gene chip data, the granulosa cells in the follicle locally produce 235 of these proteins.

Evaluation of absolute peptide quantitation strategies using selected reaction monitoring.

The use of internal peptide standards in selected reaction monitoring experiments enables absolute quantitation. Here, we describe three approaches addressing calibration of peptide concentrations in complex matrices and assess their performance in terms of trueness and precision. The simplest approach described is single reference point quantitation where a heavy peptide is spiked into test samples and the endogenous analyte quantified relative to the heavy peptide internal standard.

Simultaneous analysis of glycosylated and sialylated prostate-specific antigen revealing differential distribution of glycosylated prostate-specific antigen isoforms in prostate cancer tissues.

Aberrant protein glycosylation has been shown to be associated with disease progression and can be potentially useful as a biomarker if disease-specific glycosylation can be identified. However, high-throughput quantitative analysis of protein glycosylation derived from clinical specimens presents technical challenges due to the typically high complexity of biological samples. In this study, a mass spectrometry-based analytical method was developed to measure different glycosylated forms of glycoproteins from complex biological samples by coupling glycopeptide extraction strategy for specific glycosylation with selected reaction monitoring (SRM).

Mass spectrometric discovery and selective reaction monitoring (SRM) of putative protein biomarker candidates in first trimester Trisomy 21 maternal serum.

The accurate diagnosis of Trisomy 21 requires invasive procedures that carry a risk of miscarriage. The current state-of-the-art maternal serum screening tests measure levels of PAPP-A, free bhCG, AFP, and uE3 in various combinations with a maximum sensitivity of 60-75% and a false positive rate of 5%. There is currently an unmet need for noninvasive screening tests with high selectivity that can detect pregnancies at risk, preferably within the first trimester.

Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants.

Background: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84.

Methods: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described.

Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network: an initial study in GSC11 glioblastoma stem cells.

Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types.

Conformational flexibility, internal hydrogen bonding, and passive membrane permeability: successful in silico prediction of the relative permeabilities of cyclic peptides.

We report an atomistic physical model for the passive membrane permeability of cyclic peptides. The computational modeling was performed in advance of the experiments and did not involve the use of "training data". The model explicitly treats the conformational flexibility of the peptides by extensive conformational sampling in low (membrane) and high (water) dielectric environments.

Testing the conformational hypothesis of passive membrane permeability using synthetic cyclic peptide diastereomers.

Little is known about the effect of conformation on passive membrane diffusion rates in small molecules. Evidence suggests that intramolecular hydrogen bonding may play a role by reducing the energetic cost of desolvating hydrogen bond donors, especially amide N-H groups. We set out to test this hypothesis by investigating the passive membrane diffusion characteristics of a series of cyclic peptide diastereomers based on the sequence cyclo[Leu-Leu-Leu-Leu-Pro-Tyr].