A Mechanically Resilient Soft Hydrogel Improves Drug Delivery for Treating Post-Traumatic Osteoarthritis in Physically Active Joints.

Intra-articular delivery of disease-modifying osteoarthritis drugs (DMOADs) is likely to be most effective in early post-traumatic osteoarthritis (PTOA) when symptoms are minimal and patients are physically active. DMOAD delivery systems therefore must withstand repeated mechanical loading without affecting the drug release kinetics. Although soft materials are preferred for DMOAD delivery, mechanical loading can compromise their structural integrity and disrupt drug release. Here, we report a mechanically resilient soft hydrogel that rapidly self-heals under conditions resembling human running while maintaining sustained release of the cathepsin-K inhibitor L-006235 used as a proof-of-concept DMOAD. Notably, this hydrogel outperformed a previously reported hydrogel designed for intra-articular drug delivery, used as a control in our study, which neither recovered nor maintained drug release under mechanical loading. Upon injection into mouse knee joints, the hydrogel showed consistent release kinetics of the encapsulated agent in both treadmill-running and non-running mice. In a mouse model of aggressive PTOA exacerbated by treadmill running, L-006235 hydrogel markedly reduced cartilage degeneration. To our knowledge, this is the first hydrogel proven to withstand human running conditions and enable sustained DMOAD delivery in physically active joints, and the first study demonstrating reduced disease progression in a severe PTOA model under rigorous physical activity, highlighting the hydrogel's potential for PTOA treatment in active patients.

Characterization of the interactome profiling of Mycoplasma fermentans DnaK in cancer cells reveals interference with key cellular pathways.

Chaperone proteins are redundant in nature and, to achieve their function, they bind a large repertoire of client proteins. DnaK is a bacterial chaperone protein that recognizes misfolded and aggregated proteins and drives their folding and intracellular trafficking. Some Mycoplasmas are associated with cancers, and we demonstrated that infection with a strain of Mycoplasma fermentans isolated in our lab promoted lymphoma in a mouse model. Its DnaK is expressed intracellularly in infected cells, it interacts with key proteins to hamper essential pathways related to DNA repair and p53 functions and uninfected cells can take-up extracellular DnaK. We profile here for the first time the eukaryotic proteins interacting with DnaK transiently expressed in five cancer cell lines. A total of 520 eukaryotic proteins were isolated by immunoprecipitation and identified by Liquid Chromatography Mass Spectrometry (LC-MS) analysis. Among the cellular DnaK-binding partners, 49 were shared between the five analyzed cell lines, corroborating the specificity of the interaction of DnaK with these proteins. Enrichment analysis revealed multiple RNA biological processes, DNA repair, chromatin remodeling, DNA conformational changes, protein-DNA complex subunit organization, telomere organization and cell cycle as the most significant ontology terms. This is the first study to show that a bacterial chaperone protein interacts with key eukaryotic components thus suggesting DnaK could become a perturbing hub for the functions of important cellular pathways. Given the close interactions between bacteria and host cells in the local microenvironment, these results provide a foundation for future mechanistic studies on how bacteria interfere with essential cellular processes.

Phostensin enables lymphocyte integrin activation and population of peripheral lymphoid organs.

Rap1 GTPase drives assembly of the Mig-10/RIAM/Lamellipodin (MRL protein)-integrin-talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (Ptsn), a regulatory subunit of protein phosphatase 1, is a component of the complex. Ptsn mediates dephosphorylation of Rap1, thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes Ptsn, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable, in part, to defective activation of integrins αLß2 and α4ß7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, Ptsn enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of Ptsn ameliorates T cell-mediated colitis.

Observation of Bothrops atrox Snake Envenoming Blister Formation from Five Patients: Pathophysiological Insights.

In the Brazilian Amazon, Bothrops atrox snakebites are frequent, and patients develop tissue damage with blisters sometimes observed in the proximity of the wound. Antivenoms do not seem to impact blister formation, raising questions regarding the mechanisms underlying blister formation. Here, we launched a clinical and laboratory-based study including five patients who followed and were treated by the standard clinical protocols. Blister fluids were collected for proteomic analyses and molecular assessment of the presence of venom and antivenom. Although this was a small patient sample, there appeared to be a correlation between the time of blister appearance (shorter) and the amount of venom present in the serum (higher). Of particular interest was the biochemical identification of both venom and antivenom in all blister fluids. From the proteomic analysis of the blister fluids, all were observed to be a rich source of damage-associated molecular patterns (DAMPs), immunomodulators, and matrix metalloproteinase-9 (MMP-9), suggesting that the mechanisms by which blisters are formed includes the toxins very early in envenomation and continue even after antivenom treatment, due to the pro-inflammatory molecules generated by the toxins in the first moments after envenomings, indicating the need for local treatments with anti-inflammatory drugs plus toxin inhibitors to prevent the severity of the wounds.

Spatiotemporal Crossover between Low- and High-Temperature Dynamical Regimes in the Quantum Heisenberg Magnet.

The stranglehold of low temperatures on fascinating quantum phenomena in one-dimensional quantum magnets has been challenged recently by the discovery of anomalous spin transport at high temperatures. Whereas both regimes have been investigated separately, no study has attempted to reconcile them. For instance, the paradigmatic quantum Heisenberg spin-1/2 chain falls at low temperature within the Tomonaga-Luttinger liquid framework, while its high-temperature dynamics is superdiffusive and relates to the Kardar-Parisi-Zhang universality class in 1+1 dimensions. This Letter aims at reconciling the two regimes. Building on large-scale matrix product state simulations, we find that they are connected by a temperature-dependent spatiotemporal crossover. As the temperature T is reduced, we show that the onset of superdiffusion takes place at longer length and timescales ∝1/T. This prediction has direct consequences for experiments including nuclear magnetic resonance: it is consistent with earlier measurements on the nearly ideal Heisenberg S=1/2 chain compound Sr_{2}CuO_{3}, yet calls for new and dedicated experiments.

Androgen signaling uses a writer and a reader of ADP-ribosylation to regulate protein complex assembly.

Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

Observation of Bothrops atrox snake envenoming blister formation from five patients: pathophysiological insights

In the Brazilian Amazon, Bothrops atrox snakebites are frequent, and patients develop tissue damage with blisters sometimes observed in the proximity of the wound. Antivenoms do not seem to impact blister formation, raising questions regarding the mechanisms underlying blister formation. Here, we launched a clinical and laboratory-based study including five patients who followed and were treated by the standard clinical protocols. Blister fluids were collected for proteomic analyses and molecular assessment of the presence of venom and antivenom. Although this was a small patient sample, there appeared to be a correlation between the time of blister appearance (shorter) and the amount of venom present in the serum (higher). Of particular interest was the biochemical identification of both venom and antivenom in all blister fluids. From the proteomic analysis of the blister fluids, all were observed to be a rich source of damage-associated molecular patterns (DAMPs), immunomodulators, and matrix metalloproteinase-9 (MMP-9), suggesting that the mechanisms by which blisters are formed includes the toxins very early in envenomation and continue even after antivenom treatment, due to the pro-inflammatory molecules generated by the toxins in the first moments after envenomings, indicating the need for local treatments with anti-inflammatory drugs plus toxin inhibitors to prevent the severity of the wounds.

Characterization of a novel AraC/XylS-regulated family of N-acyltransferases in pathogens of the order Enterobacterales.

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.

LMO7 deficiency reveals the significance of the cuticular plate for hearing function.

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.

DBC1 Regulates p53 Stability via Inhibition of CBP-Dependent p53 Polyubiquitination.

The control of p53 protein stability is critical to its tumor suppressor functions. The CREB binding protein (CBP) transcriptional co-activator co-operates with MDM2 to maintain normally low physiological p53 levels in cells via exclusively cytoplasmic E4 polyubiquitination activity. Using mass spectrometry to identify nuclear and cytoplasmic CBP-interacting proteins that regulate compartmentalized CBP E4 activity, we identified deleted in breast cancer 1 (DBC1) as a stoichiometric CBP-interacting protein that negatively regulates CBP-dependent p53 polyubiquitination, stabilizes p53, and augments p53-dependent apoptosis. TCGA analysis demonstrated that solid tumors often retain wild-type p53 alleles in conjunction with DBC1 loss, supporting the hypothesis that DBC1 is selected for disruption during carcinogenesis as a surrogate for p53 functional loss. Because DBC1 maintains p53 stability in the nucleus, where p53 exerts its tumor-suppressive transcriptional function, replacement of DBC1 functionality in DBC1-deleted tumors might enhance p53 function and chemosensitivity for therapeutic benefit.

Towards improving proximity labeling by the biotin ligase BirA.

The discovery and validation of protein-protein interactions provides a knowledge base that is critical for defining protein networks and how they underpin the biology of the cell. Identification of protein interactions that are highly transient, or sensitive to biochemical disruption, can be very difficult. This challenge has been met by proximity labeling methods which generate reactive species that chemically modify neighboring proteins. The most widely used proximity labeling method is BioID, which features a mutant biotin ligase BirA(Arg118Gly), termed BirA*, fused to a protein of interest. Here, we explore how amino acid substitutions at Arg118 affect the biochemical properties of BirA. We found that relative to wild-type BirA, the Arg118Lys substitution both slightly reduced biotin affinity and increased the release of reactive biotinyl-5'-AMP. BioID using a BirA(Arg118Lys)-Lamin A fusion enabled identification of PCNA as a lamina-proximal protein in HEK293T cells, a finding that was validated by immunofluorescence microscopy. Our data expand on the concept that proximity labeling by BirA fused to proteins of interest can be modulated by amino acid substitutions that affect biotin affinity and the release of biotinyl-5'-AMP.

Author Correction: Towards an arthritis flare-responsive drug delivery system.

In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from the National Football League Players Association.

Towards an arthritis flare-responsive drug delivery system.

Local delivery of therapeutics for the treatment of inflammatory arthritis (IA) is limited by short intra-articular half-lives. Since IA severity often fluctuates over time, a local drug delivery method that titrates drug release to arthritis activity would represent an attractive paradigm in IA therapy. Here we report the development of a hydrogel platform that exhibits disassembly and drug release controlled by the concentration of enzymes expressed during arthritis flares. In vitro, hydrogel loaded with triamcinolone acetonide (TA) releases drug on-demand upon exposure to enzymes or synovial fluid from patients with rheumatoid arthritis. In arthritic mice, hydrogel loaded with a fluorescent dye demonstrates flare-dependent disassembly measured as loss of fluorescence. Moreover, a single dose of TA-loaded hydrogel but not the equivalent dose of locally injected free TA reduces arthritis activity in the injected paw. Together, our data suggest flare-responsive hydrogel as a promising next-generation drug delivery approach for the treatment of IA.

Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5+ population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2ß1, integrin ß4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP+ cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5+ ISCs. Considering the key roles Lgr5+ ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy).

Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9.

ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases either conjugate a single ADP-ribose to a target or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+-dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly(ADP-ribose) binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly(ADP-ribose) regulation of E3 activity.

Identification of actin beta-like 2 (ACTBL2) as novel, upregulated protein in colorectal cancer.

Early diagnosis of colorectal cancer (CRC) can be of value for increasing the survival rate of patients. Recently, proteomic strategies to identify markers for the diagnosis of cancer at an early stage have been employed with noteworthy results. To extend these studies, we utilized two dimensional gel electrophoresis and mass spectrometry for expression profiling of proteins extracted from the freshly frozen human colorectal cancer tissue specimens and the comparable regions of adjacent normal mucosa (serving as controls). Four gel spots were determined to be differentially stained between the tumor and the control samples on a consistent basis. Following mass spectrometric analysis of these spots, six proteins were identified; five of these had previously been reported to be associated with colorectal cancer. One protein actin beta-like 2 (ACTBL2), not linked with colorectal cancer in the earlier reports, was however found to be at higher abundance in colorectal tumor samples both by proteomics and immunohistochemistry analysis. Thus ACTBL2 association and differential upregulation in colorectal cancer is novel, and as such may contribute to our understanding of the colorectal carcinogenesis and potentially serve a function in developing markers for colorectal cancer. BIOLOGICAL SIGNIFICANCE: Colorectal cancer (CRC) is a major cause of death world-wide and good markers for early detection are lacking. In this study we conducted a proteomic analysis of tumor vs. normal tissue. We corroborated the finding of a number of previously identified proteins associated with CRC and more importantly identified a novel protein, ACTBL2, which we demonstrated to be upregulated in CRC. As additional proteins associated with CRC are identified the potential for developing panels of markers may be realized with better outcomes in early cancer detection.

Archaeal flagellin combines a bacterial type IV pilin domain with an Ig-like domain.

The bacterial flagellar apparatus, which involves ∼40 different proteins, has been a model system for understanding motility and chemotaxis. The bacterial flagellar filament, largely composed of a single protein, flagellin, has been a model for understanding protein assembly. This system has no homology to the eukaryotic flagellum, in which the filament alone, composed of a microtubule-based axoneme, contains more than 400 different proteins. The archaeal flagellar system is simpler still, in some cases having ∼13 different proteins with a single flagellar filament protein. The archaeal flagellar system has no homology to the bacterial one and must have arisen by convergent evolution. However, it has been understood that the N-terminal domain of the archaeal flagellin is a homolog of the N-terminal domain of bacterial type IV pilin, showing once again how proteins can be repurposed in evolution for different functions. Using cryo-EM, we have been able to generate a nearly complete atomic model for a flagellar-like filament of the archaeon Ignicoccus hospitalis from a reconstruction at ∼4-Å resolution. We can now show that the archaeal flagellar filament contains a ß-sandwich, previously seen in the FlaF protein that forms the anchor for the archaeal flagellar filament. In contrast to the bacterial flagellar filament, where the outer globular domains make no contact with each other and are not necessary for either assembly or motility, the archaeal flagellin outer domains make extensive contacts with each other that largely determine the interesting mechanical properties of these filaments, allowing these filaments to flex.

Proteomic identification of gender molecular markers in Bothrops jararaca venom.

Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity. BIOLOGICAL SIGNIFICANCE: Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout-vent length and weight, with females being larger than males. This sexual size dimorphism suggests the tendency for female specimens to feed on larger prey, and for male specimens to go on a diet similar to that of juveniles. Variation in the snake venom proteome is a ubiquitous phenomenon occurring at all taxonomic levels. At the intraspecific variation level, the individual contribution to the venom proteome is important but effects contributed by age and feeding habits may also affect the proteome phenotype. Whether sex-based factors play a role in venom variation of a species that shows sexual size dimorphism is poorly known. The use of proteomic strategies supported by transcriptomic data allows a more comprehensive assessment of venom proteomes uncovering components that are gender-specific.

Nonzero-temperature entanglement negativity of quantum spin models: Area law, linked cluster expansions, and sudden death.

We show that the bipartite logarithmic entanglement negativity (EN) of quantum spin models obeys an area law at all nonzero temperatures. We develop numerical linked cluster (NLC) expansions for the "area-law" logarithmic entanglement negativity as a function of temperature and other parameters. For one-dimensional models the results of NLC are compared with exact diagonalization on finite systems and are found to agree very well. The NLC results are also obtained for two dimensional XXZ and transverse field Ising models. In all cases, we find a sudden onset (or sudden death) of negativity at a finite temperature above which the negativity is zero. We use perturbation theory to develop a physical picture for this sudden onset (or sudden death). The onset of EN or its magnitude are insensitive to classical finite-temperature phase transitions, supporting the argument for absence of any role of quantum mechanics at such transitions. On approach to a quantum critical point at T=0, negativity shows critical scaling in size and temperature.

Integrative analysis to select cancer candidate biomarkers to targeted validation.

Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS.