Trauma-associated human neutrophil alterations revealed by comparative proteomics profiling.

Purpose: Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown.

Experimental Design: We applied 2D-LC-MS/MS-based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls.

Results: A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury.

Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size.

Nanoparticle biological activity, biocompatibility and fate can be directly affected by layers of readily adsorbed host proteins in biofluids. Here, we report a study on the interactions between human blood plasma proteins and nanoparticles with a controlled systematic variation of properties using (18)O-labeling and LC-MS-based quantitative proteomics. We developed a novel protocol to both simplify isolation of nanoparticle bound proteins and improve reproducibility.

Trypsin-catalyzed oxygen-18 labeling for quantitative proteomics.

Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In (18)O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with (18)O, thus providing the labeled peptide with a 4 Da mass shift from the (16)O-labeled sample.

Blood peptidome-degradome profile of breast cancer.

Background: Cancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients.

Methodology And Principal Findings: The peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP) and age, race, and menopausal status matched control healthy persons (HP) were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides.

Clinical microfluidics for neutrophil genomics and proteomics.

Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies.

Plasma proteome response to severe burn injury revealed by 18O-labeled "universal" reference-based quantitative proteomics.

A burn injury represents one of the most severe forms of human trauma and is responsible for significant mortality worldwide. Here, we present the first quantitative proteomics investigation of the blood plasma proteome response to severe burn injury by comparing the plasma protein concentrations of 10 healthy control subjects with those of 15 severe burn patients at two time-points following the injury. The overall analytical strategy for this work integrated immunoaffinity depletion of the 12 most abundant plasma proteins with cysteinyl-peptide enrichment-based fractionation prior to LC-MS analyses of individual patient samples.

Strategy for degradomic-peptidomic analysis of human blood plasma.

Herein we describe a strategy for degradomic-peptidomic analyses. The human blood peptidome was isolated through application of AC/SEC, which enriched its components by >300-fold. The isolated peptidome components were separated by long column HRLC providing a peak capacity of approximately 300 for species having MWs of up to 20 kDa.

Region-specific protein abundance changes in the brain of MPTP-induced Parkinson's disease mouse model.

Parkinson's disease (PD) is characterized by dopaminergic neurodegeneration in the nigrostriatal region of the brain; however, the neurodegeneration extends well beyond dopaminergic neurons. To gain a better understanding of the molecular changes relevant to PD, we applied two-dimensional LC-MS/MS to comparatively analyze the proteome changes in four brain regions (striatum, cerebellum, cortex, and the rest of brain) using a MPTP-induced PD mouse model with the objective to identify potential nigrostriatal-specific and other region-specific protein abundance changes. The combined analyses resulted in the identification of 4,895 nonredundant proteins with at least two unique peptides per protein.

Endogenous 3,4-dihydroxyphenylalanine and dopaquinone modifications on protein tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical.

Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3,4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first proteome survey of endogenous site-specific modifications, i.

N-terminal enrichment: developing a protocol to detect specific proteolytic fragments.

Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a "bottom-up" strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments.

Mouse-specific tandem IgY7-SuperMix immunoaffinity separations for improved LC-MS/MS coverage of the plasma proteome.

We report on a mouse specific SuperMix immunoaffinity separation system for separating low-abundance proteins from high and moderate abundance proteins in mouse plasma. When applied in tandem with a mouse IgY7 column that removes the seven most abundant proteins in plasma, the SuperMix column captures more than 100 additional moderate abundance proteins, thus allowing significant enrichment of low-abundance proteins in the flow-through fraction. A side-by-side comparison of results obtained from 2D-LC-MS/MS analyses of flow-through samples from IgY7 and SuperMix columns revealed a nearly 2-fold improvement in the overall proteome coverage.

Global systems-level analysis of Hfq and SmpB deletion mutants in Salmonella: implications for virulence and global protein translation.

Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages.

A simple procedure for effective quenching of trypsin activity and prevention of 18O-labeling back-exchange.

Trypsin-catalyzed stable isotope 16O/18O-labeling of the C-terminal carboxyl groups of peptides is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by residual trypsin that can catalyze the back-exchange of 18O with 16O after labeling. Here, we demonstrate through a detailed evaluation that boiling the peptide sample for 10 min provides a simple means for completely quenching residual trypsin activity and preventing oxygen back-exchange in 18O-labeled samples.

Large-scale multiplexed quantitative discovery proteomics enabled by the use of an (18)O-labeled "universal" reference sample.

The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope (18)O-labeled "universal" reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the (18)O-labeled "universal" reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on (16)O/(18)O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample.

Enhanced detection of low abundance human plasma proteins using a tandem IgY12-SuperMix immunoaffinity separation strategy.

The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids such as blood plasma, which is an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates approximately 60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction.

Dynamically multiplexed ion mobility time-of-flight mass spectrometry.

Ion mobility spectrometry-time-of-flight mass spectrometry (IMS-TOFMS) has been increasingly used in analysis of complex biological samples. A major challenge is to transform IMS-TOFMS to a high-sensitivity, high-throughput platform, for example, for proteomics applications. In this work, we have developed and integrated three advanced technologies, including efficient ion accumulation in an ion funnel trap prior to IMS separation, multiplexing (MP) of ion packet introduction into the IMS drift tube, and signal detection with an analog-to-digital converter, into the IMS-TOFMS system for the high-throughput analysis of highly complex proteolytic digests of, for example, blood plasma.