MatrisomeDB 2.0: 2023 updates to the ECM-protein knowledge database.

The extracellular matrix (ECM) is a complex assembly of proteins that constitutes the scaffold organizing cells, tissues, and organs. Over the past decade, mass-spectrometry-based proteomics has become the method of choice to profile the composition of the ECM, or the matrisome, of tissues. To assist non-specialists with the reuse of ECM proteomic datasets, we released MatrisomeDB (https://matrisomedb.org) in 2020. Here, we report the expansion of the database to include 25 new curated studies on the ECM of 24 new tissues in addition to datasets on tissues previously included, more than doubling the size of the original database and achieving near-complete coverage of the in-silico predicted matrisome. We further enhanced data visualization by maps of peptides and post-translational-modifications detected onto domain-based representations and 3D structures of ECM proteins. We also referenced external resources to facilitate the design of targeted mass spectrometry assays. Last, we implemented an abstract-mining tool that generates an enrichment word cloud from abstracts of studies in which a queried protein is found with higher confidence and higher abundance relative to other studies in MatrisomeDB.

MatrisomeDB: the ECM-protein knowledge database.

The extracellular matrix (ECM) is a complex and dynamic meshwork of cross-linked proteins that supports cell polarization and functions and tissue organization and homeostasis. Over the past few decades, mass-spectrometry-based proteomics has emerged as the method of choice to characterize the composition of the ECM of normal and diseased tissues. Here, we present a new release of MatrisomeDB, a searchable collection of curated proteomic data from 17 studies on the ECM of 15 different normal tissue types, six cancer types (different grades of breast cancers, colorectal cancer, melanoma, and insulinoma) and other diseases including vascular defects and lung and liver fibroses. MatrisomeDB (http://www.pepchem.org/matrisomedb) was built by retrieving raw mass spectrometry data files and reprocessing them using the same search parameters and criteria to allow for a more direct comparison between the different studies. The present release of MatrisomeDB includes 847 human and 791 mouse ECM proteoforms and over 350 000 human and 600 000 mouse ECM-derived peptide-to-spectrum matches. For each query, a hierarchically-clustered tissue distribution map, a peptide coverage map, and a list of post-translational modifications identified, are generated. MatrisomeDB is the most complete collection of ECM proteomic data to date and allows the building of a comprehensive ECM atlas.

A Novel Truncated Form of Serum Amyloid A in Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD) is an acute vasculitis in children that can cause coronary artery abnormalities. Its diagnosis is challenging, and many cytokines, chemokines, acute phase reactants, and growth factors have failed evaluation as specific biomarkers to distinguish KD from other febrile illnesses. We performed protein profiling, comparing plasma from children with KD with febrile control (FC) subjects to determine if there were specific proteins or peptides that could distinguish the two clinical states. MATERIALS AND METHODS: Plasma from three independent cohorts from the blood of 68 KD and 61 FC subjects was fractionated by anion exchange chromatography, followed by surface-enhanced laser desorption ionization (SELDI) mass spectrometry of the fractions. The mass spectra of KD and FC plasma samples were analyzed for peaks that were statistically significantly different. RESULTS: A mass spectrometry peak with a mass of 7,860 Da had high intensity in acute KD subjects compared to subacute KD (p = 0.0003) and FC (p = 7.9 x 10-10) subjects. We identified this peak as a novel truncated form of serum amyloid A with N-terminal at Lys-34 of the circulating form and validated its identity using a hybrid mass spectrum immunoassay technique. The truncated form of serum amyloid A was present in plasma of KD subjects when blood was collected in tubes containing protease inhibitors. This peak disappeared when the patients were examined after their symptoms resolved. Intensities of this peptide did not correlate with KD-associated laboratory values or with other mass spectrum peaks from the plasma of these KD subjects. CONCLUSIONS: Using SELDI mass spectrometry, we have discovered a novel truncated form of serum amyloid A that is elevated in the plasma of KD when compared with FC subjects. Future studies will evaluate its relevance as a diagnostic biomarker and its potential role in the pathophysiology of KD.

Genomic compartmentalization of gene families encoding core components of metazoan signaling systems.

To investigate the role of gene localization and genome organization in cell-cell signalling and regulation, we mapped the distribution pattern of gene families that comprise core components of intercellular communication networks. Our study is centered on the distinct evolutionarily conserved metazoan signalling pathways that employ proteins in the receptor tyrosine kinase, WNT, hedgehog, NOTCH, Janus kinase/STAT, transforming growth factor beta, and nuclear hormone receptor protein families. Aberrant activity of these signalling pathways is closely associated with the promotion and maintenance of human cancers. The cataloguing and mapping of genes encoding these signalling proteins and comparisons across species has led us to propose that the genome can be subdivided into six genome-wide primary linkage groups (PLGs). PLGs are composed of assemblages of gene families that are often mutually exclusive, raising the possibility of unique functional identities for each group. Examination of the localization patterns of genes with distinct functions in signal transduction demonstrates dichotomous segregation patterns. For example, gene families of cell-surface receptors localize to genomic compartments that are distinct from the locations of their cognate ligand gene families. Additionally, genes encoding negative-acting components of signalling pathways (inhibitors and antagonists) are topologically separated from their positive regulators and other signal transducer genes. We, therefore, propose the existence of conserved genomic territories that encode key proteins required for the proper activity of metazoan signaling and regulatory systems. Disruption in this pattern of topologic genomic organization may contribute to aberrant regulation in hereditary or acquired diseases such as cancer. We further propose that long-range looping genomic regulatory interactions may provide a mechanism favouring the remarkable retention of these conserved gene clusters during chordate evolution.

Alterations in cerebrospinal fluid proteins in a presymptomatic primary glioma model.

BACKGROUND: Understanding the early relationship between brain tumor cells and their environment could lead to more sensitive biomarkers and new therapeutic strategies. We have been using a rodent model of neurocarcinogenesis in which all animals develop brain tumors by six months of age to establish two early landmarks in glioma development: the appearance of a nestin(+) cell at thirty days of age and the appearance of cellular hyperplasia between 60 and 120 days of age. We now report an assessment of the CSF proteome to determine the changes in protein composition that occur during this period. MATERIALS AND METHODS: Nestin(+) cell clusters and microtumors were assessed in 63 ethylnitrosourea-exposed rats on 30, 60, and 90 days of age. CSF was obtained from the cisterna magna from 101 exposed and control rats at 30, 60, and 90 days and then analyzed using mass spectrometry. Differentially expressed peaks were isolated and identified. RESULTS: Nestin(+) cells were noted in all ethylnitrosourea-exposed rats assessed pathologically. Small microtumors were noted in 0%, 18%, and 67% of 30-, 60-, and 90-day old rats, respectively (p<0.05, Chi square). False Discovery Rate analysis of peak intensities showed that the number of true discoveries with p<0.05 increased markedly with increasing age. Isolation and identification of highly differentially detected proteins at 90 days of age revealed increases in albumin and a fragment of α1 macroglobulin and alterations in glutathionylated transthyretin. CONCLUSIONS: The presence of increased albumin, fragments of cerebrospinal fluid proteins, and glutathione breakdown in temporal association with the development of cellular hyperplasia, suggests that, similar to many other systemic cancers, inflammation and oxidative stress is playing an important early role in the host's response to brain tumor development and may be involved in affecting the early growth of brain tumor.

Identification of protein marker in vaginal wall tissues of women with stress urinary incontinence by protein chip array.

AIM: We sought to investigate protein biomarkers for stress urinary incontinence (SUI) in vaginal tissues using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and examine if this is a reliable methodology to examine proteins in small tissue specimens. MATERIAL AND METHODS: We compared protein expression profile of vaginal tissue from women with SUI and continent controls. A 22.6kDa peak was identified by subsequent weak cation-exchange, reverse-phase fractionation, gel electrophoresis, and trypsin digestion, then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and MALDI MS-MS. Biomarker identity and expression level were confirmed by Western-blotting and immunohistochemistry. RESULTS: Expression of the 22.6kDa protein, identified as SM-22α, was significantly higher in women with SUI versus controls. A 3×3-mm tissue sample was sufficient for identification. Western-blot/immunohistochemistry confirmed the SELDI-TOS MS findings. CONCLUSION: SM-22α, a marker for myofibroblasts, was identified as a biomarker of SUI. Differential protein profiling by SELDI-TOF MS is a powerful and reliable tool for urogynecological research as it allows us to study an array of proteins simultaneously using small tissue samples.

A diagnostic algorithm combining clinical and molecular data distinguishes Kawasaki disease from other febrile illnesses.

BACKGROUND: Kawasaki disease is an acute vasculitis of infants and young children that is recognized through a constellation of clinical signs that can mimic other benign conditions of childhood. The etiology remains unknown and there is no specific laboratory-based test to identify patients with Kawasaki disease. Treatment to prevent the complication of coronary artery aneurysms is most effective if administered early in the course of the illness. We sought to develop a diagnostic algorithm to help clinicians distinguish Kawasaki disease patients from febrile controls to allow timely initiation of treatment. METHODS: Urine peptidome profiling and whole blood cell type-specific gene expression analyses were integrated with clinical multivariate analysis to improve differentiation of Kawasaki disease subjects from febrile controls. RESULTS: Comparative analyses of multidimensional protein identification using 23 pooled Kawasaki disease and 23 pooled febrile control urine peptide samples revealed 139 candidate markers, of which 13 were confirmed (area under the receiver operating characteristic curve (ROC AUC 0.919)) in an independent cohort of 30 Kawasaki disease and 30 febrile control urine peptidomes. Cell type-specific analysis of microarrays (csSAM) on 26 Kawasaki disease and 13 febrile control whole blood samples revealed a 32-lymphocyte-specific-gene panel (ROC AUC 0.969). The integration of the urine/blood based biomarker panels and a multivariate analysis of 7 clinical parameters (ROC AUC 0.803) effectively stratified 441 Kawasaki disease and 342 febrile control subjects to diagnose Kawasaki disease. CONCLUSIONS: A hybrid approach using a multi-step diagnostic algorithm integrating both clinical and molecular findings was successful in differentiating children with acute Kawasaki disease from febrile controls.

Effects of moderate versus deep hypothermic circulatory arrest and selective cerebral perfusion on cerebrospinal fluid proteomic profiles in a piglet model of cardiopulmonary bypass.

OBJECTIVE: Our objective was to compare protein profiles of cerebrospinal fluid between control animals and those subjected to cardiopulmonary bypass after moderate versus deep hypothermic circulatory arrest with selective cerebral perfusion. METHODS: Immature Yorkshire piglets were assigned to one of four study groups: (1) deep hypothermic circulatory arrest at 18 degrees C, (2) deep hypothermic circulatory arrest at 18 degrees C with selective cerebral perfusion, (3) moderate hypothermic circulatory arrest at 25 degrees C with selective cerebral perfusion, or (4) age-matched control animals without surgery. Animals undergoing cardiopulmonary bypass were cooled to their assigned group temperature and exposed to 1 hour of hypothermic circulatory arrest. After arrest, animals were rewarmed, weaned off bypass, and allowed to recover for 4 hours. Cerebrospinal fluid collected from surgical animals after the recovery period was compared with cerebrospinal fluid from controls by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Protein spectra were analyzed for differences between groups by Mann-Whitney U test and false discovery rate analysis. RESULTS: Baseline and postbypass physiologic parameters were similar in all surgical groups. A total of 194 protein peaks were detected. Compared with controls, groups 1, 2, and 3 had 64, 100, and 13 peaks that were significantly different, respectively (P < .05). Three of these peaks were present in all three groups. Cerebrospinal fluid protein profiles in animals undergoing cardiopulmonary bypass with moderate hypothermic circulatory arrest (group 3) were more similar to controls than either of the groups subjected to deep hypothermia. CONCLUSIONS: The mass spectra of cerebrospinal fluid proteins are altered in piglets exposed to cardiopulmonary bypass and hypothermic circulatory arrest. Moderate hypothermic circulatory arrest (25 degrees C) with selective cerebral perfusion compared with deep hypothermic circulatory arrest (18 degrees C) is associated with fewer changes in cerebrospinal fluid proteins, when compared with nonbypass controls.

Plasma biomarkers in a mouse model of preterm labor.

Preterm labor (PTL) is frequently associated with inflammation. We hypothesized that biomarkers during pregnancy can identify pregnancies most at risk for development of PTL. An inflammation-induced mouse model of PTL was used. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to analyze and compare the plasma protein (PP) profile between CD-1 mice injected intrauterine with either lipopolysaccharide (LPS) or PBS on d 14.5 of gestation. The median differences of normalized PP peaks between the two groups were determined using the Mann-Whitney U test and the false discovery rate. In a second series of experiments, both groups of mice were injected with a lower dose of LPS. A total of 1665 peaks were detected. Thirty peaks were highly differentially expressed (p < 0.0001) between the groups. Two 11 kDa protein peaks were identified by MALDI-TOF/TOF-MS and confirmed to be mouse serum amyloid A (SAA) 1 and 2. Plasma SAA2 levels were increased in LPS-treated animals compared with controls and in LPS-treated animals that delivered preterm vs. those that delivered at term. SAA2 has the potential to be a plasma biomarker that can identify pregnancies at risk for development of PTL.

Capture of phosphopeptides using alpha-zirconium phosphate nanoplatelets.

Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. Alpha-ZrPN were able specifically to capture phosphoserine-containing peptides from a tryptic digest of a complex peptide mixture in which its abundance was only 0.05%. Alpha-ZrPN also bound peptides containing phosphothreonine and phosphotyrosine. The limit of detection for phosphopeptides is approximately 2 fmol, based on using matrix-assisted laser desorption/ionization mass spectrometry. Alpha-ZrPN were applied for the analysis of tryptic digests of mouse liver and leukemia cell phosphoproteomes and succeeded in identifying 158 phosphopeptides (209 phosphorylation sites) from 101 phosphoproteins in mouse liver lysate and 78 phosphopeptides (104 phosphorylation sites) from 59 phosphoproteins in leukemia cell extract. For these two tryptic digests, the alpha-ZrPN approach is able to capture more phosphopeptides than that obtained from TiO2 particles or from Fe(3+)-IMAC beads, but each method is able to bind some phosphopeptides that the others do not.

A potential biomarker in the cord blood of preterm infants who develop retinopathy of prematurity.

Preterm infants are at risk of developing sepsis, necrotizing enterocolitis (NEC), chronic lung disease (CLD), and retinopathy of prematurity (ROP). We used high-throughput mass spectrometry to investigate whether cord blood proteins can be used to predict development of these morbidities. Cord blood plasma from 44 infants with a birth weight of <1500 g was analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). Six infants developed ROP >or=stage II, 10 CLD, three sepsis, and one NEC. We detected 814 protein signals representing 330 distinct protein species. Nineteen biomarkers were associated with development of >or=stage II ROP [false-discovery rate (FDR) <5%] and none with CLD. Several proteins with molecular weight (Mr) 15-16 kD and pI 4-5 were detected with increased abundance in infants with ROP, while similar Mr proteins with pI 7-9 were less abundant in these patients. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sequence analysis identified these proteins as alpha-, beta-, and gamma-globin chains. Partial deamidation of Asn139 in beta-globin chains was observed only in the pI 4-5 proteins. We conclude that there are several promising biomarkers for the risk of ROP. Deamidation of globin chains is especially promising and may indicate underlying prenatal pathologic mechanisms in ROP. Validation studies will be undertaken to determine their clinical utility.