Vocal fold tissue failure: preliminary data and constitutive modeling

In human voice production (phonation), linear small-amplitude vocal fold oscillation occurs only under restricted conditions. Physiologically, phonation more often involves large-amplitude oscillation associated with tissue stresses and strains beyond their linear viscoelastic limits, particularly in the lamina propria extracellular matrix (ECM). This study reports some preliminary measurements of tissue deformation and failure response of the vocal fold ECM under large-strain shear The primary goal was to formulate and test a novel constitutive model for vocal fold tissue failure, based on a standard-linear cohesive-zone (SL-CZ) approach. Tissue specimens of the sheep vocal fold mucosa were subjected to torsional deformation in vitro, at constant strain rates corresponding to twist rates of 0.01, 0.1, and 1.0 rad/s. The vocal fold ECM demonstrated nonlinear stress-strain and rate-dependent failure response with a failure strain as low as 0.40 rad. A finite-element implementation of the SL-CZ model was capable of capturing the rate dependence in these preliminary data, demonstrating the model's potential for describing tissue failure. Further studies with additional tissue specimens and model improvements are needed to better understand vocal fold tissue failure.     

The predicted secretomes of Monosiga brevicollis and Capsaspora owczarzaki,close unicellular relatives of metazoans,reveal new insights into the evolution of the metazoan extracellular matrix

The extracellular matrix (ECM) is a major mediator of multi-cellularity in the metazoa. Multiple ECM proteins are conserved from sponges to human, raising questions about the evolutionary origin of ECM. Choanoflagellates are the closest unicellular relatives of the metazoa and proteins with domains characteristic of metazoan ECM proteins have been identified from the genome-predicted proteome of the choanoflagellate Monosiga brevicollis. However, a systematic analysis of M. brevicollis secretory signal peptide-containing proteins with ECM domains has been lacking. We analysed all predicted secretory signal-peptide-containing proteins of M. brevicollis for ECM domains. Nine domains that are widespread in metazoan ECM proteins are represented, with EGF, fibronectin III, laminin G, and von Willebrand Factor_A domains being the most numerous. Three proteins contain more than one category of ECM domain, however, no proteins correspond to the domain architecture of metazoan ECM proteins. The fibronectin III domains are all present within glycoside hydrolases and none contain an integrin-binding motif. Glycosaminoglycan-binding motifs identified in animal thrombospondin type 1 domains are conserved in some M. brevicollis representatives of this domain, whereas there is little evidence of conservation of glycosaminoglycan-binding motifs in the laminin G domains. The identified proteins were compared with the predicted secretory ECM domain-containing proteins of the integrin-expressing filasterean, Capsaspora owczarzaki. C. owczarzaki encodes a smaller number of secretory, ECM domain-containing proteins and only EGF, fibronectin type III and laminin G domains are represented. The M. brevicollis and C. owczarzaki proteins have distinct domain architectures and all proteins differ in their domain architecture to metazoan ECM proteins. These identifications provide a basis for future experiments to validate the extracellular location of these proteins and uncover their functions in choanoflagellates and C. owczarzaki. The data strengthen the model that ECM proteins are metazoan-specific and evolved as innovations in the last common metazoan ancestor.     

Application of peptide LC retention time information in a discriminant function for peptide identification by tandem mass spectrometry

We describe the application of a peptide retention time reversed phase liquid chromatography (RPLC) prediction model previously reported (Petritis et al. Anal. Chem. 2003, 75, 1039) for improved peptide identification. The model uses peptide sequence information to generate a theoretical (predicted) elution time that can be compared with the observed elution time. Using data from a set of known proteins, the retention time parameter was incorporated into a discriminant function for use with tandem mass spectrometry (MS/MS) data analyzed with the peptide/protein identification program SEQUEST. For singly charged ions, the number of confident identifications increased by 12% when the elution time metric is included compared to when mass spectral data is the sole source of information in the context of a Drosophila melanogaster database. A 3-4% improvement was obtained for doubly and triply charged ions for the same biological system. Application to the larger Rattus norvegicus (rat) and human proteome databases resulted in an 8-9% overall increase in the number of confident identifications, when both the discriminant function and elution time are used. The effect of adding "runner-up" hits (peptide matches that are not the highest scoring for a spectra) from SEQUEST is also explored, and we find that the number of confident identifications is further increased by 1% when these hits are also considered. Finally, application of the discriminant functions derived in this work with approximately 2.2 million spectra from over three hundred LC-MS/MS analyses of peptides from human plasma protein resulted in a 16% increase in confident peptide identifications (9022 vs 7779) using elution time information. Further improvements from the use of elution time information can be expected as both the experimental control of elution time reproducibility and the predictive capability are improved.     

Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues

Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. In this study, we evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis (CITP)-based proteomic platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues. In comparison to our previous studies utilizing capillary isoelectric focusing, the CITP-based analysis is more robust and increases proteome coverage. In this case, results from three FFPE liver tissues yield a total of 4098 distinct Swiss-Prot identifications at a 1% false-discovery rate. To judge the accuracy of these assignments, immunohistochemistry is performed on a panel of 17 commonly assayed proteins. These proteins span a wide range of protein abundances as inferred from relative quantitation via spectral counting. Among the panel were 4 proteins identified by a single peptide hit, including three clusters of differentiation (CD) markers: CD74, CD117, and CD45. Because single peptide hits are often regarded with skepticism, it is notable that all proteins tested by IHC stained positive.     

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.     

Approaches in extracellular matrix engineering for determination of adhesion molecule mediated single cell function

The native extracellular matrix (ECM) and the cells that comprise human tissues are together engaged in a complex relationship; cells alter the composition and structure of the ECM to regulate the material and biologic properties of the surrounding environment while the composition and structure of the ECM modulates cellular processes that maintain healthy tissue and repair diseased tissue. This reciprocal relationship occurs via cell adhesion molecules (CAMs) such as integrins, selectins, cadherins and IgSF adhesion molecules. To study these cell-ECM interactions, researchers use two-dimensional substrates or three-dimensional matrices composed of native proteins or bioactive peptide sequences to study single cell function. While two-dimensional substrates provide valuable information about cell-ECM interactions, three-dimensional matrices more closely mimic the native ECM; cells cultured in three-dimensional matrices have demonstrated greater cell movement and increased integrin expression when compared to cells cultured on two-dimensional substrates. In this article we review a number of cellular processes (adhesion, motility, phagocytosis, differentiation and survival) and examine the cell adhesion molecules and ECM proteins (or bioactive peptide sequences) that mediate cell functionality.     

Changes in expression of extracellular matrix genes, fibrogenic factors, and actin cytoskeletal organization in retinol treated and untreated vocal fold stellate cells.

The regulation of extracellular matrix (ECM) constituency is critical in maintaining vocal cord biomechanical viscoelasticity required for phonation. Recently our laboratory successfully isolated and cultured a novel cell called a vocal fold stellate cell (VFSC), thought to play a central role in laryngeal ECM metabolism, aging, scarring and cancer. Our laboratory has shown that these cells undergo transdifferentiation that is partially reversed by exposure to all-trans retinol (ATROH). Here we make the first report on the expression of various ECM components, MMPs, TIMPs, pro-fibrogenic cytokines, and other ECM modulators in transdifferentiated and deactivated VFSCs. We show that VFSCs maintain an ECM expression pattern similar to laryngeal cancer and scars but distinct from tracheal fibroblasts. Exposure to ATROH differentially affects the VFSC expression of ECM components, matrix-regulating enzymes, and fibrogenic factors suggesting that the inhibitory effects of this synthetic cofactor should be studied further in laryngeal fibrosis and scarring. We also show that increased exposure to retinol induces sequential reorganization of the actin cytoskeleton in activated VFSCs. Our findings demonstrate that VFSCs are capable of regulating vocal fold ECM constituency important throughout normal laryngeal development. Furthermore, our results implicate VFSC activation in ECM misregulation which is a hallmark of several laryngeal pathologies.     

Gel based isoelectric focusing of peptides and the utility of isoelectric point in protein identification

Here we present the theoretical and experimental evaluation of peptide isoelectric point as a method to aid in the identification of peptides from complex mixtures. Predicted pI values were found to match closely the experimentally obtained data, resulting in the development of a unique filter that lowers the effective false positive rate for peptide identification. Due to the reduction of the false positive rate, the cross-correlation parameters Xcorr and deltaCn from the SEQUEST program can be lowered resulting in 25% more peptide identifications. This approach was successfully applied to analysis of the soluble fraction of the E. coli proteome, where 417 proteins were identified from 1022 peptides using just 20 microg of material.     

Proteins associated with the Myxococcus xanthus extracellular matrix

Fruiting body formation of Myxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT), fibA, and pilA backgrounds. Disruption of the MXAN4860 gene had no effect in the WT or fibA background but in the pilA background resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that the M. xanthus ECM proteome is diverse and novel.     

Expanding the zebrafish embryo proteome using multiple fractionation approaches and tandem mass spectrometry

The proteome of zebrafish, Danio rerio, embryos has not been studied in great detail mainly due to the presence of high abundance yolk proteins in embryos. Here we report the highest number of the zebrafish embryo proteins identified so far to our knowledge, through a combination of a protein-level fractionation approach (1D SDS-PAGE) and two different peptide-level fractionation approaches (IEF and strong anion exchange (SAX)) of deyolked zebrafish embryos followed by LC-MS/MS. We detected 5267 proteins in total of which 3464 proteins were identified with at least two peptides (less than 1% peptide false discovery rate). The analysis of proteome coverage from each method showed that 56% of detected proteins were common to all approaches and 95% of the detected proteome was obtained from 1D SDS-PAGE approach alone. Bioinformatics analysis of the detected proteome demonstrated that nucleocytoplasmic transport (biological process) and ribosomal proteins (cellular component) were the most over-represented proteins, whereas cell-cell signaling (biological process) and extracellular space proteins (cellular component) were the most under-represented proteins in the identified proteome.     

Extracellular matrix proteome of chickpea (Cicer arietinum L.) illustrates pathway abundance, novel protein functions and evolutionary perspect

The extracellular matrix (ECM) or cell wall is a dynamic system and serves as the first line mediator in cell signaling to perceive and transmit extra- and intercellular signals in many pathways. Although ECM is a conserved compartment ubiquitously present throughout evolution, a compositional variation does exist among different organisms. ECM proteins account for 10% of the ECM mass, however, comprise several hundreds of different molecules with diverse functions. To understand the function of ECM proteins, we have developed the cell wall proteome of a crop legume, chickpea (Cicer arietinum). This comprehensive overview of the proteome would provide a basis for future comparative proteomic efforts for this important crop. Proteomic analyses revealed new ECM proteins of unknown functions vis-à-vis the presence of many known cell wall proteins. In addition, we report here evidence for the presence of unexpected proteins with known biochemical activities, which have never been associated with ECM.     

Comprehensive yeast proteome analysis using a capillary isoelectric focusing-based multidimensional separation platform coupled with ESI-MS/MS

As demonstrated in this study, a CIEF-based multidimensional separation platform not only is compatible with the detergent-based membrane protein preparation protocol, but also achieves both the largest yeast membrane proteome coverage and the most comprehensive analysis of the yeast proteome to date. By using a 1% false discovery rate for total peptide identifications, a total of 2513 distinct yeast proteins are identified from the SDS-solubilized fraction with an average of 5.4 peptides leading to each protein identification. Among proteins identified from the SDS-solubilized fraction, 407 proteins are predicted to contain at least two or more transmembrane domains using TMHMM (www.cbs.dtu.dk/services/TMHMM-2.0/), corresponding to 46% yeast membrane proteome coverage. Only four additional membrane proteins are identified in the soluble and urea-solubilized fractions, affirming the utility of SDS extraction for enriching the membrane proteome. By combining proteome results obtained from the soluble, urea-solubilized, and SDS-solubilized fractions, a single yeast proteome analysis yields the identification of 3632 distinct yeast proteins, corresponding to 55% theoretical yeast proteome coverage or 70% of proteins predicted to be expressed during log-phase growth in rich media.     

An accurate mass tag strategy for quantitative and high-throughput proteome measurements

We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide "accurate mass tags" (AMTs) produced by global protein enzymatic digestion. The two-stage strategy exploits Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from "potential mass tags" identified using conventional tandem mass spectrometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FT-ICR measurements has been shown capable of characterizing peptide mixtures of significantly more than 10(5) components with mass accuracies of < 1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian proteomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for >60% of the potentially expressed proteins in the organism Deinococcus radiodurans.     

Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis: the human proteome

Large-scale protein identifications from highly complex protein mixtures have recently been achieved using multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) and subsequent database searching with algorithms such as SEQUEST. Here, we describe a probability-based evaluation of false positive rates associated with peptide identifications from three different human proteome samples. Peptides from human plasma, human mammary epithelial cell (HMEC) lysate, and human hepatocyte (Huh)-7.5 cell lysate were separated by strong cation exchange (SCX) chromatography coupled offline with reversed-phase capillary LC-MS/MS analyses. The MS/MS spectra were first analyzed by SEQUEST, searching independently against both normal and sequence-reversed human protein databases, and the false positive rates of peptide identifications for the three proteome samples were then analyzed and compared. The observed false positive rates of peptide identifications for human plasma were significantly higher than those for the human cell lines when identical filtering criteria were used, suggesting that the false positive rates are significantly dependent on sample characteristics, particularly the number of proteins found within the detectable dynamic range. Two new sets of filtering criteria are proposed for human plasma and human cell lines, respectively, to provide an overall confidence of >95% for peptide identifications. The new criteria were compared, using a normalized elution time (NET) criterion (Petritis et al. Anal. Chem. 2003, 75, 1039-1048), with previously published criteria (Washburn et al. Nat. Biotechnol. 2001, 19, 242-247). The results demonstrate that the present criteria provide significantly higher levels of confidence for peptide identifications from mammalian proteomes without greatly decreasing the number of identifications.     

Dehydration-responsive reversible and irreversible changes in the extracellular matrix: comparative proteomics of chickpea genotypes with contrasting tolerance

Dehydration is the most crucial environmental factor that limits plant growth, development, and productivity affecting agriculture throughout the world. Studies on genetic variations for dehydration tolerance in plants is crucial because divergent cultivars with contrasting traits aid the identification of key cellular components that confer better adaptability. The extracellular matrix (ECM) is a dynamic structure that serves as the repository for important signaling components and acts as a front-line defense. To better understand dehydration adaptation, a proteomic study was performed on the extracellular matrix of ICCV-2, a dehydration-susceptible genotype of chickpea. The proteome was generated with ECM-enriched fractions using two-dimensional gel electrophoresis. The LC-ESI-MS/MS analysis led to the identification of 81 dehydration-responsive proteins. The proteome was then compared with that of JG-62, a tolerant genotype. Comparative proteomics revealed genotype-specific expression of many proteins involved in a variety of cellular functions. Further, the reversible and irreversible changes in the proteomes revealed their differing ability to recover from dehydration-induced damage. We propose that cell wall restructuring and superior homeostasis, particularly the management of reactive oxygen species, may render better dehydration-adaptation. To our knowledge, this is the first report on the comprehensive comparison of dehydration-responsive organellar proteome of two genotypes with contrasting tolerance.     

A Novel Cell Fixation Method that Greatly Enhances Protein Identification in Microproteomic Studies Using Laser Capture Microdissection and Mass Spectrometry

Microproteomic studies have improved our knowledge of cell biology. Yet, with mass spectrometry (MS) analysis, accuracy can be lost for protein identification and quantification when using heterogeneous samples. Laser capture microdissection (LCM) allows for the enrichment of specific subsets of cells to study their proteome; however, sample fixation is necessary. Unfortunately, fixation hampers MS results due to protein cross‐linking. The aim of this study was to identify both a fixation protocol and an extraction method that returns the best yield of proteins for downstream MS analysis, while preserving cellular structures. We compared glutaraldehyde (GLU), a common fixative to preserve cells, to dithiobispropionimidate (DTBP), a cleavable cross‐linker. Our DTBP fixation/extraction protocol greatly increased the protein recovery. In fact, while 1000 GLU fixed cells returned only 159 unique protein hits, from 1464 unique peptides of 1994 unique collected spectra, 1000 DTBP fixed cells resulted in 567 unique collected protein hits, from 7542 unique peptides, of 10,401 unique collected spectra. That is, a 3.57‐fold increase in protein hits, 5.15‐fold increase in unique peptides, and a 5.22‐fold increase in unique collected spectra. Overall, the novel protocol introduced here allows for a very efficient protein recovery and good sample quality for MS after sample collection using LCM.     

Effect of 2MEGA labeling on membrane proteome analysis using LC-ESI QTOF MS

One of the challenges associated with large-scale proteome analysis using tandem mass spectrometry (MS/MS) and automated database searching is to reduce the number of false positive identifications without sacrificing the number of true positives found. In this work, a systematic investigation of the effect of 2MEGA labeling (N-terminal dimethylation after lysine guanidination) on the proteome analysis of a membrane fraction of an Escherichia coli cell extract by 2-dimensional liquid chromatography MS/MS is presented. By a large-scale comparison of MS/MS spectra of native peptides with those from the 2MEGA-labeled peptides, the labeled peptides were found to undergo facile fragmentation with enhanced a1 or a1-related (a(1)-17 and a(1)-45) ions derived from all N-terminal amino acids in the MS/MS spectra; these ions are usually difficult to detect in the MS/MS spectra of nonderivatized peptides. The 2MEGA labeling alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI and ESI MS experiments. 2MEGA labeling was found not only to increase the number of peptides and proteins identified but also to generate enhanced a1 or a1-related ions as a constraint to reduce the number of false positive identifications. In total, 640 proteins were identified from the E. coli membrane fraction, with each protein identified based on peptide mass and sequence match of one or more peptides using MASCOT database search algorithm from the MS/MS spectra generated by a quadrupole time-of-flight mass spectrometer. Among them, the subcellular locations of 336 proteins are presently known, including 258 membrane and membrane-associated proteins (76.8%). Among the classified proteins, there was a dramatic increase in the total number of integral membrane proteins identified in the 2MEGA-labeled sample (153 proteins) versus the unlabeled sample (77 proteins).