COI1-dependent jasmonate signalling affects growth, metabolite production and cell wall protein composition in arabidopsis.

Background and Aims: Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described. Methods: Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels. Key Results: Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as ß-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses. Conclusions: This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.

Recruitment of septin cytoskeletal proteins by botulinum toxin A protease determines its remarkable stability.

Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.

Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy.

Abnormal assemblies formed by misfolded superoxide dismutase-1 (SOD1) proteins are the likely cause of SOD1-linked familial amyotrophic lateral sclerosis (fALS) and may be involved in some cases of sporadic ALS. To analyze the structure of the insoluble SOD1 amyloid fibrils, we first used limited proteolysis followed by mass spectrometric analysis. Digestion of amyloid fibrils formed from full-length N-acetylated WT SOD1 with trypsin, chymotrypsin, or Pronase revealed that the first 63 residues of the N terminus were protected from protease digestion by fibril formation. Furthermore, every tested ALS-mutant SOD1 protein (G37R, L38V, G41D, G93A, G93S, and D101N) showed a similar protected fragment after trypsin digestion. Our second approach to structural characterization used atomic force microscopy to image the SOD1 fibrils and revealed that WT and mutants showed similar twisted morphologies. WT fibrils had a consistent average helical pitch distance of 62.1 nm. The ALS-mutant SOD1 proteins L38V, G93A, and G93S formed fibrils with helical twist patterns very similar to those of WT, whereas small but significant structural deviations were observed for the mutant proteins G37R, G41D, and D101N. Overall, our studies suggest that human WT SOD1 and ALS-mutants tested have a common intrinsic propensity to fibrillate through the N terminus and that single amino acid substitutions can lead to changes in the helical twist pattern.

Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.

The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

Molecular dissection of novel trafficking and processing of the Toxoplasma gondii rhoptry metalloprotease toxolysin-1.

Toxoplasma gondii utilizes specialized secretory organelles called rhoptries to invade and hijack its host cell. Many rhoptry proteins are proteolytically processed at a highly conserved SΦXE site to remove organellar targeting sequences that may also affect protein activity. We have studied the trafficking and biogenesis of a secreted rhoptry metalloprotease with homology to insulysin that we named toxolysin-1 (TLN1). Through genetic ablation and molecular dissection of TLN1, we have identified the smallest rhoptry targeting domain yet reported and expanded the consensus sequence of the rhoptry pro-domain cleavage site. In addition to removal of its pro-domain, TLN1 undergoes a C-terminal cleavage event that occurs at a processing site not previously seen in Toxoplasma rhoptry proteins. While pro-domain cleavage occurs in the nascent rhoptries, processing of the C-terminal region precedes commitment to rhoptry targeting, suggesting that it is mediated by a different maturase, and we have identified residues critical for proteolysis. We have additionally shown that both pieces of TLN1 associate in a detergent-resistant complex, formation of which is necessary for trafficking of the C-terminal portion to the rhoptries. Together, these studies reveal novel processing and trafficking events that are present in the protein constituents of this unusual secretory organelle.

A peroxidase-dependent apoplastic oxidative burst in cultured Arabidopsis cells functions in MAMP-elicited defense.

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

mzML--a community standard for mass spectrometry data.

Mass spectrometry is a fundamental tool for discovery and analysis in the life sciences. With the rapid advances in mass spectrometry technology and methods, it has become imperative to provide a standard output format for mass spectrometry data that will facilitate data sharing and analysis. Initially, the efforts to develop a standard format for mass spectrometry data resulted in multiple formats, each designed with a different underlying philosophy. To resolve the issues associated with having multiple formats, vendors, researchers, and software developers convened under the banner of the HUPO PSI to develop a single standard. The new data format incorporated many of the desirable technical attributes from the previous data formats, while adding a number of improvements, including features such as a controlled vocabulary with validation tools to ensure consistent usage of the format, improved support for selected reaction monitoring data, and immediately available implementations to facilitate rapid adoption by the community. The resulting standard data format, mzML, is a well tested open-source format for mass spectrometer output files that can be readily utilized by the community and easily adapted for incremental advances in mass spectrometry technology.

Mass spectrometry (LC-MS/MS) identified proteomic biosignatures of breast cancer in proximal fluid.

We have begun an early phase of biomarker discovery in three clinically important types of breast cancer using a panel of human cell lines: HER2 positive, hormone receptor positive and HER2 negative, and triple negative (HER2-, ER-, PR-). We identified and characterized the most abundant secreted, sloughed, or leaked proteins released into serum free media from these breast cancer cell lines using a combination of protein fractionation methods before LC-MS/MS mass spectrometry analysis. A total of 249 proteins were detected in the proximal fluid of 7 breast cancer cell lines. The expression of a selected group of high abundance and/or breast cancer-specific potential biomarkers including thromobospondin 1, galectin-3 binding protein, cathepsin D, vimentin, zinc-α2-glycoprotein, CD44, and EGFR from the breast cancer cell lines and in their culture media were further validated by Western blot analysis. Interestingly, mass spectrometry identified a cathepsin D protein single-nucleotide polymorphism (SNP) by alanine to valine replacement from the MCF-7 breast cancer cell line. Comparison of each cell line media proteome displayed unique and consistent biosignatures regardless of the individual group classifications, demonstrating the potential for stratification of breast cancer. On the basis of the cell line media proteome, predictive Tree software was able to categorize each cell line as HER2 positive, HER2 negative, and hormone receptor positive and triple negative based on only two proteins, muscle fructose 1,6-bisphosphate aldolase and keratin 19. In addition, the predictive Tree software clearly identified MCF-7 cell line overexpresing the HER2 receptor with the SNP cathepsin D biomarker.

Epithelial junctions depend on intercellular trans-interactions between the Na,K-ATPase ß1 subunits.

N-Glycans of the Na,K-ATPase ß1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the ß1 subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous ß1 subunit with various YFP-linked ß1 subunits expressed in Madin-Darby canine kidney cells was used to assess ß1-ß1 interactions. The amount of co-precipitated endogenous dog ß1 was greater with dog YFP-ß1 than with rat YFP-ß1, showing that amino acid-mediated interactions are important for ß1-ß1 binding. Co-precipitation of ß1 was also less with the unglycosylated YFP-ß1 than with glycosylated YFP-ß1, indicating a role for N-glycans. Mixing cells expressing dog YFP-ß1 with non-transfected cells increased the amount of co-precipitated ß1, confirming the presence of intercellular (YFP-ß1)-ß1 complexes. Accordingly, disruption of intercellular junctions decreased the amount of co-precipitated ß1 subunits. The decrease in ß1 co-precipitation both with rat YFP-ß1 and unglycosylated YFP-ß1 was associated with decreased detergent stability of junctional proteins and increased paracellular permeability. Reducing N-glycan branching by specific inhibitors increased (YFP-ß1)-ß1 co-precipitation and strengthened intercellular junctions. Therefore, interactions between the ß1 subunits of neighboring cells maintain integrity of intercellular junctions, and alterations in the ß1 subunit N-glycan structure can regulate stability and tightness of intercellular junctions.

Phenotypic and genomic analysis of hypervirulent human-associated Bordetella bronchiseptica.

BACKGROUND: B. bronchiseptica infections are usually associated with wild or domesticated animals, but infrequently with humans. A recent phylogenetic analysis distinguished two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Complex IV isolates appear to have a bias for infecting humans; however, little is known regarding their epidemiology, virulence properties, or comparative genomics. RESULTS: Here we report a characterization of the virulence of human-associated complex IV B. bronchiseptica strains. In in vitro cytotoxicity assays, complex IV strains showed increased cytotoxicity in comparison to a panel of complex I strains. Some complex IV isolates were remarkably cytotoxic, resulting in LDH release levels in A549 cells that were 10- to 20-fold greater than complex I strains. In vivo, a subset of complex IV strains was found to be hypervirulent, with an increased ability to cause lethal pulmonary infections in mice. Hypercytotoxicity in vitro and hypervirulence in vivo were both dependent on the activity of the bsc T3SS and the BteA effector. To clarify differences between lineages, representative complex IV isolates were sequenced and their genomes were compared to complex I isolates. Although our analysis showed there were no genomic sequences that can be considered unique to complex IV strains, there were several loci that were predominantly found in complex IV isolates. CONCLUSION: Our observations reveal a T3SS-dependent hypervirulence phenotype in human-associated complex IV isolates, highlighting the need for further studies on the epidemiology and evolutionary dynamics of this B. bronchiseptica lineage.

Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry.

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation.

Deconvolution and database search of complex tandem mass spectra of intact proteins: a combinatorial approach.

Top-down proteomics studies intact proteins, enabling new opportunities for analyzing post-translational modifications. Because tandem mass spectra of intact proteins are very complex, spectral deconvolution (grouping peaks into isotopomer envelopes) is a key initial stage for their interpretation. In such spectra, isotopomer envelopes of different protein fragments span overlapping regions on the m/z axis and even share spectral peaks. This raises both pattern recognition and combinatorial challenges for spectral deconvolution. We present MS-Deconv, a combinatorial algorithm for spectral deconvolution. The algorithm first generates a large set of candidate isotopomer envelopes for a spectrum, then represents the spectrum as a graph, and finally selects its highest scoring subset of envelopes as a heaviest path in the graph. In contrast with other approaches, the algorithm scores sets of envelopes rather than individual envelopes. We demonstrate that MS-Deconv improves on Thrash and Xtract in the number of correctly recovered monoisotopic masses and speed. We applied MS-Deconv to a large set of top-down spectra from Yersinia rohdei (with a still unsequenced genome) and further matched them against the protein database of related and sequenced bacterium Yersinia enterocolitica. MS-Deconv is available at http://proteomics.ucsd.edu/Software.html.

Post-translational modifications of integral membrane proteins resolved by top-down Fourier transform mass spectrometry with collisionally activated dissociation.

Integral membrane proteins remain a challenge to proteomics because they contain domains with physicochemical properties poorly suited to today's bottom-up protocols. These transmembrane regions may potentially contain post-translational modifications of functional significance, and thus development of protocols for improved coverage in these domains is important. One way to achieve this goal is by using top-down mass spectrometry whereby the intact protein is subjected to mass spectrometry and dissociation. Here we describe top-down high resolution Fourier transform mass spectrometry with collisionally activated dissociation to study post-translationally modified integral membrane proteins with polyhelix bundle and transmembrane porin motifs and molecular masses up to 35 kDa. On-line LC-MS analysis of the bacteriorhodopsin holoprotein yielded b- and y-ions that covered the full sequence of the protein and cleaved 79 of 247 peptide bonds (32%). The experiment proved that the mature sequence consists of residues 14-261, confirming N-terminal propeptide cleavage and conversion of N-terminal Gln-14 to pyrrolidone carboxylic acid (-17.02 Da) and C-terminal removal of Asp-262. Collisionally activated dissociation fragments localized the N(6)-(retinylidene) modification (266.20 Da) between residues 225-248 at Lys-229, the sole available amine in this stretch. Off-line nanospray of all eight subunits of the cytochrome b(6)f complex from the cyanobacterium Nostoc PCC 7120 defined various post-translational modifications, including covalently attached c-hemes (615.17 Da) on cytochromes f and b. Analysis of murine mitochondrial voltage-dependent anion channel established the amenability of the transmembrane beta-barrel to top-down MS and localized a modification site of the inhibitor Ro 68-3400 at Cys-232. Where neutral loss of the modification is a factor, only product ions that carry the modification should be used to assign its position. Although bond cleavage in some transmembrane alpha-helical domains was efficient, other regions were refractory such that their primary structure could only be inferred from the coincidence of genomic translation with precursor and product ions that spanned them.

NdhP and NdhQ: two novel small subunits of the cyanobacterial NDH-1 complex.

The subunit composition of the NAD(P)H dehydrogenase complex of Thermosynechococcus elongatus was analyzed by different types of mass spectrometry. All 15 known subunits (NdhA-NdhO) were identified in the purified NDH-1L complex. Moreover, two additional intact mass tags of 4902.7 and 4710.5 Da could be assigned after reannotation of the T. elongatus genome. NdhP and NdhQ are predicted to contain a single transmembrane helix each, and homologues are apparent in other cyanobacteria. Additionally, ndhP is present in some cyanophages in a cluster of PSI genes and exhibits partial similarity to NDF6, a subunit of the plant NDH-1 complex.

A novel 3-methylhistidine modification of yeast ribosomal protein Rpl3 is dependent upon the YIL110W methyltransferase.

We have shown that Rpl3, a protein of the large ribosomal subunit from baker's yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven ß-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.

Amide bonds assemble pili on the surface of bacilli.

Pilin precursors are the building blocks of pili on the surface of Gram-positive bacteria; however, the assembly mechanisms of these adhesive fibers are unknown. Here, we describe the chemical bonds that assemble BcpA pilin subunits on the surface of Bacillus cereus. Sortase D cleaves BcpA precursor between the threonine (T) and the glycine (G) residues of its LPXTG sorting signal and catalyzes formation of an amide bond between threonine (T) of the sorting signal and lysine (K) in the YPKN motif of another BcpA subunit. Three CNA B domains of BcpA generate intramolecular amide bonds, and one of these contributes also to pilus formation. Conservation of catalysts and structural elements in pilin precursors in Gram-positive bacteria suggests a universal mechanism of fiber assembly.

Data-directed top-down Fourier-transform mass spectrometry of a large integral membrane protein complex: photosystem II from Galdieria sulphuraria.

High-resolution top-down MS was used to characterize eleven integral and five peripheral subunits of the 750 kDa photosystem II complex from the eukaryotic red alga, Galdieria sulphuraria. The primary separation used LC MS with concomitant fraction collection (LC-MS+), yielding around 40 intact mass tags at 100 ppm mass accuracy on a low-resolution ESI mass spectrometer, whose retention and mass were used to guide subsequent high-resolution top-down nano-electrospray FT ion-cyclotron resonance MS experiments (FT-MS). Both collisionally activated and electron capture dissociation were used to confirm the presence of eleven small subunits to mass accuracy within 5 ppm; PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbT, PsbX and PsbZ. All subunits showed covalent modifications that fall into three classes including retention of initiating formyl-methionine, removal of methionine at the N-terminus with or without acetylation, and removal of a longer N-terminal peptide. Peripheral subunits identified by top-down analysis included oxygen-evolving complex subunits PsbO, PsbU, PsbV, as well as Psb28 (PsbW) and Psb27 ("PsbZ-like"). Top-down high-resolution MS provides the necessary precision, typically less than 5 ppm, for identification and characterization of polypeptide composition of these important membrane protein complexes.

The minimum information about a proteomics experiment (MIAPE).

Both the generation and the analysis of proteomics data are now widespread, and high-throughput approaches are commonplace. Protocols continue to increase in complexity as methods and technologies evolve and diversify. To encourage the standardized collection, integration, storage and dissemination of proteomics data, the Human Proteome Organization's Proteomics Standards Initiative develops guidance modules for reporting the use of techniques such as gel electrophoresis and mass spectrometry. This paper describes the processes and principles underpinning the development of these modules; discusses the ramifications for various interest groups such as experimentalists, funders, publishers and the private sector; addresses the issue of overlap with other reporting guidelines; and highlights the criticality of appropriate tools and resources in enabling 'MIAPE-compliant' reporting.

Quantitative Proteomics Using Formalin-fixed, Paraffin-embedded Biopsy Tissues in Inflammatory Disease.

BACKGROUND: Investigations in human disease pathogenesis have been hampered due to paucity of access to fresh-frozen tissues (FFT) for use in global, data-driven methodologies. As an alternative, formalin-fixed, paraffin-embedded (FFPE) tissues are readily available in pathology banks. However, the use of formalin for fixation can lead to the loss of proteins that appear during inflammation, thus introducing an inherent sample bias. To address this, we compared FF and FFPE tissue proteomics to determine whether FFPE-tissue can be used effectively in inflammatory diseases. METHODS: Adjacent kidney slices from lupus nephritic mice were processed as FFPE or FFTs. Their tissue lysates were run together using proteomics workflow involving filter-aided sample preparation, in-solution dimethyl isotope labeling, StageTip fractionation, and nano-LC MS/MS through an Orbitrap XL MS. RESULTS: We report a >97% concordance in protein identification between adjacent FFPE and FFTs in murine lupus nephritic kidneys. Specifically, proteins representing pathways, namely, 'systemic lupus erythematosus', 'interferon-α', 'TGF-ß', and 'extracellular matrix', were reproducibly quantified between FFPE and FFTs. However, 12%-29% proteins were quantified differently in FFPE compared to FFTs, but the differences were consistent across experiments. In particular, certain proteins represented in pathways, including 'inflammatory response' and 'innate immune system' were quantified less in FFPE than in FFTs. In a pilot study of human FFPE tissues, we identified proteins relevant to pathogenesis in lupus nephritic kidney biopsies compared to control kidneys. CONCLUSION: This is the first report of lupus nephritis kidney proteomics using FFPE tissue. We concluded that archived FFPE tissues can be reliably used for proteomic analyses in inflammatory diseases, with a caveat that certain proteins related to immunity and inflammation may be quantified less in FFPE than in FFTs.

Mass spectral analysis of the apolipoproteins on mouse high density lipoproteins. Detection of post-translational modifications.

Using mass spectrometry, we have recently reported on molecular masses of the apolipoproteins associated with porcine and equine HDL. In addition to obtaining accurate masses for the various apolipoproteins, we also were able to detect mass variations due to post-translational modifications. In the present study, we have used these same approaches to characterize the apolipoproteins in two inbred mouse strains, C57BL/6 and BALB/c. Comparing our molecular mass data with calculated values for molecular weight, we were able to identify the correct sequences for several of the major apolipoproteins. Analyses were carried out on the apolipoproteins of ultracentrifugally isolated HDL. Prior to analyses by electrospray ionization mass spectrometry (ESI-MS), the apolipoproteins were separated either by size exclusion or reverse phase chromatography. The molecular masses of apoA-I, proapoA-I, apoA-II, proapoA-II, apoC-I and apoC-III were obtained. Comparing the values obtained for the two strains, differences in the molecular masses of apoA-I, apoA-II and apoC-III were observed. In this study, post-translationally modified apolipoproteins, involving loss of amino acids from both the N- and C-termini, oxidation of methionine residues and possible acylation, were noted following reverse-phase separation. Further analyses by tandem mass spectrometry (MSMS) done on the tryptic digests of apolipoproteins separated by reverse phase chromatography enabled us to confirm sequence differences between the two strains, to verify selected apoA-I sequences that had been entered into the GenBank and to identify which methionines in apoA-I, apoC-III and apoE had been converted to methionine sulfoxides.