Retention Time Prediction for TMT-Labeled Peptides in Proteomic LC-MS Experiments.

We present the first detailed study of chromatographic behavior of peptides labeled with tandem mass tags (TMT and TMTpro) in 2D LC for proteomic applications. Carefully designed experimental procedures have permitted generating data sets of over 100,000 nonlabeled and TMT-labeled peptide pairs for the low pH RP in the second separation dimension and data sets of over 10,000 peptide pairs for high-pH RP, HILIC (amide and silica), and SCX separations in the first separation dimension. The average increase in peptide RPLC (0.

In-depth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry.

Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 μg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS).

Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT.

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study.

MicroRNA abundance is altered in synaptoneurosomes during prion disease.

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions.

Automating mass spectrometry-based quantitative glycomics using aminoxy tandem mass tag reagents with SimGlycan.

Protein glycosylation is a common post-translational modification, which serves critical roles in the biological processes of organisms. Monitoring of changes in the abundance and structure of glycans may be necessary to explain the correlations between protein glycosylation and various diseases. Hence, the growing importance of glycoproteomics necessitates in-depth qualitative and quantitative studies of glycans.

Diversity within the O-linked protein glycosylation systems of acinetobacter species.

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii.

The chromosome-centric human proteome project: a call to action.

The grand vision of the human proteome project (HPP) is moving closer to reality with the recent announcement by HUPO of the creation of the HPP consortium in charge of the development of a two-part HPP, one focused on the description of proteomes of biological samples or related to diseases (B/D-HPP) and the other dedicated to a systematic description of proteins as gene products encoded in the human genome (the C-HPP). This new initiative of HUPO seeks to identify and characterize at least one representative protein from every gene, create a protein distribution atlas and a protein pathway or network map. This vision for proteomics can be the roadmap of biological and clinical research for years to come if it delivers on its promises.

Increasing the productivity of glycopeptides analysis by using higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation.

Currently, glycans are attracting attention from the scientific community as potential biomarkers or as posttranslational modifications (PTMs) of therapeutic proteins. However, structural characterization of glycoproteins and glycopeptides remains analytically challenging. Here, we report on the implementation of a novel acquisition strategy termed higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation (HCD-PD-ETD) on a hybrid linear ion trap-orbitrap mass spectrometer.

A simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano LC coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry.

In this report we describe an on-column method for glycopeptide enrichment with cellulose as a solid-phase extraction material. The method was developed using tryptic digests of several standard glycoproteins and validated with more complex standard protein digest mixtures. Glycopeptides of different masses containing neutral and acidic glycoforms of both N- and O-linked sugars were obtained in good yield by this method.

Enhanced sensitivity in proteomics experiments using FAIMS coupled with a hybrid linear ion trap/Orbitrap mass spectrometer.

We describe the use and application of high-field asymmetric waveform ion mobility spectrometry combined with nanoscale liquid chromatography mass spectrometry (nanoLC-FAIMS-MS) to improve the sensitivity and dynamic range of proteomics analyses on a hybrid LTQ-Orbitrap mass spectrometer. The ability of FAIMS to enrich multiply protonated peptides against background ions confers a marked advantage in proteomics analyses by decreasing the limits of detection to facilitate the identification of low-abundance peptide ions. These multiply charged ions are recorded into separate acquisition channels to enhance the overall population of detectable peptide ions from a single analysis.

Proteomic mapping of stimulus-specific signaling pathways involved in THP-1 cells exposed to Porphyromonas gingivalis or its purified components.

Periodontitis is an inflammatory disease initiated by host-parasite interactions which contributes to connective tissue destruction and alveolar bone resorption. Porphyromonas gingivalis (P.g.

Application of the StrOligo algorithm for the automated structure assignment of complex N-linked glycans from glycoproteins using tandem mass spectrometry.

Oligosaccharides associated with proteins are known to give these molecules specific conformations and functions. Analysis of proteins would not be complete without studying the glycans. However, high-throughput techniques in proteomics will soon overwhelm the current capacity of methods if no automation is incorporated into glycomics.

Automated structural assignment of derivatized complex N-linked oligosaccharides from tandem mass spectra.

Glycoprotein function is controlled by several biological factors, one of them being the structure of carbohydrate chains (glycans) attached to specific amino acids of the protein backbone. Changes in glycan structures have been shown to modify the secondary and tertiary conformation of glycoproteins, thus their function. Powerful analytical tools are available for the characterization of sugar structures, and recently mass spectrometry (MS) has been increasingly useful for this purpose.

A study of immunoglobulin G glycosylation in monoclonal and polyclonal species by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.

N-linked oligosaccharides were released from human and bovine polyclonal immunoglobulin G (IgG) obtained from commercial sources and also from a monoclonal IgG(1) secreted by murine B-lymphocyte hybridoma cells (CC9C10) grown under different serum-free conditions. These conditions differed according to their steady-state dissolved oxygen concentrations. This work is based on a previous quantitative study where released glycans were characterized by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (J.