John D. Venable

A quantitative analysis software tool for mass spectrometry–based proteomics

We describe Census, a quantitative software tool compatible with many labeling strategies as well as with label-free analyses, single-stage mass spectrometry (MS1) and tandem mass spectrometry (MS/MS) scans, and high- and low-resolution mass spectrometry data. Census uses robust algorithms to address poor-quality measurements and improve quantitative efficiency, and it can support several input file formats. We tested Census with stable-isotope labeling analyses as well as label-free analyses.

Characterization of Global Yeast Quantitative Proteome Data Generated from the Wild-Type and Glucose Repression Saccharomyces cerevisiae Strains: The Comparison of Two Quantitative Methods

The quantitative proteomic analysis of complex protein mixtures is emerging as a technically challenging but viable systems-level approach for studying cellular function. This study presents a large-scale comparative analysis of protein abundances from yeast protein lysates derived from both wild-type yeast and yeast strains lacking key components of the Snf1 kinase complex. Four different strains were grown under well-controlled chemostat conditions. Multidimensional protein identification technology followed by quantitation using either spectral counting or stable isotope labeling approaches was used to identify relative changes in the protein expression levels between the strains. A total of 2388 proteins were relatively quantified, and more than 350 proteins were found to have significantly different expression levels between the two strains of comparison when using the stable isotope labeling strategy. The stable isotope labeling based quantitative approach was found to be highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found, the major reason behind the discrepancy was the lack of reproducible sampling for proteins with low spectral counts. The functional categorization of the relative protein expression differences that occur in Snf1-deficient strains uncovers a wide range of biological processes regulated by this important cellular kinase.

ProLuCID: An improved SEQUEST-like algorithm with enhanced sensitivity and specificity

ProLuCID, a new algorithm for peptide identification using tandem mass spectrometry and protein sequence databases has been developed. This algorithm uses a three tier scoring scheme. First, a binomial probability is used as a preliminary scoring scheme to select candidate peptides. The binomial probability scores generated by ProLuCID minimize molecular weight bias and are independent of database size. A modified cross-correlation score is calculated for each candidate peptide identified by the binomial probability. This cross-correlation scoring function models the isotopic distributions of fragment ions of candidate peptides which ultimately results in higher sensitivity and specificity than that obtained with the SEQUEST XCorr. Finally, ProLuCID uses the distribution of XCorr values for all of the selected candidate peptides to compute a Z score for the peptide hit with the highest XCorr. The ProLuCID Z score combines the discriminative power of XCorr and DeltaCN, the standard parameters for assessing the quality of the peptide identification using SEQUEST, and displays significant improvement in specificity over ProLuCID XCorr alone. ProLuCID is also able to take advantage of high resolution MS/MS spectra leading to further improvements in specificity when compared to low resolution tandem MS data. A comparison of filtered data searched with SEQUEST and ProLuCID using the same false discovery rate as estimated by a target-decoy database strategy, shows that ProLuCID was able to identify as many as 25% more proteins than SEQUEST. ProLuCID is implemented in Java and can be easily installed on a single computer or a computer cluster. This article is part of a Special Issue entitled: Computational Proteomics.

Identification of the anti-inflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis

Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 were identified by MS analyses using MALDI (matrix-assisted laser-desorption-ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)-tandem MS and multidimensional protein identification technology. Mutations of Ser197, Ser218 and Ser228 to alanine in the human protein significantly increased TTPs gel mobility (likely to be stoichiometric), whereas mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labelling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) respectively. The present study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.

Metalloendoprotease cleavage triggers gelsolin amyloidogenesis

Amyloid diseases like Alzheimers disease and familial amyloidosis of Finnish type (FAF) stem from endoproteolytic cleavage of a precursor protein to generate amyloidogenic peptides that accumulate as amyloid deposits in a tissue‐specific manner. FAF patients deposit both 8 and 5 kDa peptides derived from mutant (D187Y/N) plasma gelsolin in the extracellular matrix (ECM). The first of two aberrant sequential proteolytic events is executed by furin to yield a 68 kDa (C68) secreted fragment. We now identify the metalloprotease MT1‐matrix metalloprotease (MMP), an integral membrane protein active in the ECM, as a protease that processes C68 to the amyloidogenic peptides. We further demonstrate that ECM components are capable of accelerating gelsolin amyloidogenesis. Proteolysis by MT1‐MMP‐like proteases proximal to the unique chemical environment of the ECM offers an explanation for the tissue‐specific deposition observed in FAF and provides critical insight into new therapeutic strategies.

Comparison of Different Signal Thresholds on Data Dependent Sampling in Orbitrap and LTQ Mass Spectrometry for the Identification of Peptides and Proteins in Complex Mixtures

We evaluate the effect of ion-abundance threshold settings for data-dependent acquisition on a hybrid LTQ-Orbitrap mass spectrometer, analyzing features such as the total number of spectra collected, the signal to noise ratio of the full MS scans, the spectral quality of the tandem mass spectra acquired, and the number of peptides and proteins identified from a complex mixture. We find that increasing the threshold for data-dependent acquisition generally decreases the quantity but increases the quality of the spectra acquired. This is especially true when the threshold setting is set above the noise level of the full MS scan. We compare two distinct experimental configurations: one where full MS scans are acquired in the Orbitrap analyzer while tandem MS scans are acquired in the LTQ analyzer, and one where both full MS and tandem MS scans are acquired in the LTQ analyzer. We examine the number of spectra, peptides, and proteins identified under various threshold conditions, and we find that the optimal threshold setting is at or below the respective noise level of the instrument regardless of whether the full MS scan is performed in the Orbitrap or in the LTQ analyzer. When comparing the high-throughput identification performance of the two analyzers, we conclude that, used at optimal threshold levels, the LTQ and the Orbitrap identify similar numbers of peptides and proteins. The higher scan speed of the LTQ, which results in more spectra being collected, is roughly compensated by the higher mass accuracy of the Orbitrap, which results in improved database searching and peptide validation software performance.

Subzero temperature chromatography for reduced back-exchange and improved dynamic range in amide hydrogen/deuterium exchange mass spectrometry.

Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified.

Signal enhancements produced from externally generated ‘carrier’ particles in electrothermal vaporization-inductively coupled plasma mass spectrometry ☆

Abstract An externally generated carbon particle aerosol produced from the pyrolysis of hexane vapor was introduced into an electrothermal vaporization inductively coupled plasma mass spectrometer (ETV-ICPMS) system in order to probe possible ‘carrier’ effects. Signal enhancements were seen upon the introduction of the externally generated aerosol for most metals studied. However, these enhancements were determined to be caused by changes in the plasma rather than increased transport from the ETV to the ICPMS. Because a pyrolyzed hydrocarbon was used to generate the aerosol, both hydrogen gas and carbon were introduced into the plasma. The source of the enhancements is believed to stem from an increased plasma temperature (from H 2(g) addition) which increases the kinetic energy of the ions and enhances the transmission through the ion lenses. The carbon may also contribute to improved ionization efficiencies for those analytes with ionization potentials between 8.5 and 11 eV. Thus, the addition of the externally generated aerosol did produce signal enhancements, but the effect is believed to be unrelated to analyte transport and instead dependent upon changes in bulk properties of the plasma.

Isotope Coded Labeling for Accelerated Protein Interaction Profiling using MS

Protein interaction surface mapping using MS is widely applied but comparatively resource-intensive. Here, a workflow adaptation for use of isotope-coded tandem mass tags for the purpose is reported. The key benefit of improved throughput derived from sample acquisition multiplexing and automated analysis is shown to be maintained in the new application. Mapping of the epitopes of two monoclonal antibodies on their respective targets serves to illustrate the novel approach. We conclude that the approach enables mapping of interactions by MS at significantly larger scales than hereto possible.

Multiplexed electrothermal vaporization sample introduction system for inductively coupled plasma spectrometry

Abstract A multiplexed electrothermal vaporization (ETV) system for sample introduction into an inductively coupled plasma was designed in an effort to increase sample turn-around time. Tungsten filaments (300 W), originally designed for overhead projectors, were chosen for use as ETVs to avoid the high power requirements associated with other ETV devices, e.g. graphite furnaces (2–3 kW). In short, we have multiplexed the thermal stages have been multiplexed such that a vaporization event can take place every 20 s. This represents a significant increase in the throughput typically associated with ETV-ICPMS, which is normally approximately 20–30 samples/h. Evaluated with respect to common figure of merit criteria, the performance of the multiplexed ETV system is similar to that seen with conventional graphite furnace ETV systems. However, several mass spectral interferences can be introduced by the presence of W into the plasma, which hinder the analysis of certain metals (Hg, Mo, etc.). Thus, other low power vaporizers (e.g. Re, Ta) should be considered for use in future systems.