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Additional method details, tables and figures to support text in the manuscript. Figure S.1 Protein yield from LCM of regions of interest. Figure S.2 Variations on data analysis in the Perseus software. Figure S.3 Incidence of Biochemical Recurrence in Sample Cohort for Immunohistochemistry. Figure S.4 Expression of significant proteins in 'normal' patient biopsy tissue. Table S1.1 Significantly changing proteins between G3 epithelia and stroma. Table S1.2 Significantly changing proteins between G4 epithelia and stroma. Table S.1.3 Significantly changing proteins between G3 and G4 epithelia Table S1.4 Significantly changing proteins between G3 and G4 Stroma

Full list of proteins identified from LC-MS/MS analysis of all tumour regions of interest

Prostate cancer is the second most common cancer in men worldwide. Gleason grading is an important predictor of prostate cancer outcomes and is influential in determining patient treatment options. Clinical decisions based on a Gleason score of 7 are difficult as the prognosis for individuals diagnosed with Gleason 4+3 cancer is much worse than for those diagnosed with Gleason 3+4 cancer. Laser capture microdissection (LCM) is a highly precise method to isolate specific cell populations or discrete microregions from tissues. This report undertook a detailed molecular characterization of the tumor microenvironment in prostate cancer to define the proteome in the epithelial and stromal regions from tumor foci of Gleason grades 3 and 4. Tissue regions of interest were isolated from several Gleason 3+3 and Gleason 4+4 tumors using telepathology to leverage specialized pathology expertise to support LCM. Over 2,000 proteins were identified following liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of all regions of interest. Statistical analysis revealed significant differences in protein expression (>100 proteins) between Gleason 3 and Gleason 4 regions—in both stromal and epithelial compartments. A subset of these proteins has had prior strong association with prostate cancer, thereby providing evidence for the authenticity of the approach. Finally, validation of these proteins by immunohistochemistry has been obtained using an independent cohort of prostate cancer tumor specimens.Implications: This unbiased strategy provides a strong foundation for the development of biomarker protein panels with significant diagnostic and prognostic potential. Mol Cancer Res; 15(3); 281–93. ©2017 AACR.

Oral squamous cell carcinoma (OSCC) is the eight most common malignancy worldwide with an incidence rate of 40% in south-east Asia. Lack of effective diagnostic tools at early stage and disease recurrence despite extensive treatments are main reasons for high mortality and low survival rates. The aim of current study was to identify differentially expressed proteins to explore potential candidate biomarkers having diagnostic significance. We performed comparative proteomic analysis of paired protein samples (cancerous buccal mucosa and adjacent normal tissue) from OSCC patients using a combination of two dimensional gel electrophoresis and Mass spectrometric analysis. On the basis of spot intensity, seventeen proteins were found to be consistently differentially expressed among most of the samples which were identified through mass spectrometry. For validation of identified proteins, expression level of stratifin was determined using immuno-histochemistry and Western blot analysis. All identified proteins were analyzed by STRING to explore their interaction. Among uniquely identified proteins in this study, at least two candidate markers (Ig Kappa chain C region and Isoform 2 of fructose bisphosphate aldolase A) were found to be novel with respect to OSCC which can be explored further. Results presented in current study are likely to contribute in understanding the involvement of these molecules in carcinogenesis apart from their plausible role as diagnostic/prognostic markers.

Prostate cancer is the second most common cancer in men worldwide. Gleason grading is an important predictor of prostate cancer outcomes and is influential in determining patient treatment options. Clinical decisions based on a Gleason score of 7 are difficult as the prognosis for individuals diagnosed with Gleason 4+3 cancer is much worse than for those diagnosed with Gleason 3+4 cancer. Laser capture microdissection (LCM) is a highly precise method to isolate specific cell populations or discrete microregions from tissues. This report undertook a detailed molecular characterization of the tumor microenvironment in prostate cancer to define the proteome in the epithelial and stromal regions from tumor foci of Gleason grades 3 and 4. Tissue regions of interest were isolated from several Gleason 3+3 and Gleason 4+4 tumors using telepathology to leverage specialized pathology expertise to support LCM. Over 2,000 proteins were identified following liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of all regions of interest. Statistical analysis revealed significant differences in protein expression (>100 proteins) between Gleason 3 and Gleason 4 regions—in both stromal and epithelial compartments. A subset of these proteins has had prior strong association with prostate cancer, thereby providing evidence for the authenticity of the approach. Finally, validation of these proteins by immunohistochemistry has been obtained using an independent cohort of prostate cancer tumor specimens. Implications: This unbiased strategy provides a strong foundation for the development of biomarker protein panels with significant diagnostic and prognostic potential. Mol Cancer Res; 15(3); 281–93. ©2017 AACR.

When compared with hormonal therapy alone, treatment with combined hormone and radiation therapy (CHRT) gives improved disease-specific survival outcomes for patients with prostate cancer; however, a significant number of CHRT patients still succumb to recurrent disease. The purpose of this study was to use longitudinal patient samples obtained as part of an ongoing noninterventional clinical trial (ICORG06-15) to identify and evaluate a potential serum protein signature of disease recurrence. Label-free LC-MS/MS based protein discovery was undertaken on depleted serum samples from CHRT patients who showed evidence of disease recurrence (n = 3) and time-matched patient controls (n = 3). A total of 104 proteins showed a significant change between these two groups. Multiple reaction monitoring (MRM) assays were designed for a subset of these proteins as part of a panel of putative prostate cancer biomarkers (41 proteins) for evaluation in longitudinal serum samples. These data revealed significant interpatient variability in individual protein expression between time of diagnosis, disease recurrence, and beyond and serve to highlight the importance of longitudinal patient samples for evaluating the use of candidate protein biomarkers in disease monitoring.

The age-related loss of skeletal muscle mass and associated progressive decline in contractile strength is a serious pathophysiological issue in the elderly. In order to investigate global changes in the skeletal muscle proteome after the fifth decade of life, this study analysed total extracts from human vastus lateralis muscle by fluorescence difference in-gel electrophoresis. Tissue specimens were derived from middle-aged (47-62 years) vs. aged (76-82 years) individuals and potential changes in the protein expression profiles were compared between these two age groups by a comprehensive gel electrophoresis-based survey. Age-dependent alterations in the concentration of 19 protein spots were revealed and mass spectrometry identified these components as being involved in the excitation-contraction-relaxation cycle, muscle metabolism, ion handling and the cellular stress response. This indicates a generally perturbed protein expression pattern in senescent human muscle. Increased levels of mitochondrial enzymes and isoform switching of the key contractile protein, actin, support the idea of glycolytic-to-oxidative and fast-to-slow transition processes during muscle aging. Importantly, the carbonic anhydrase (CA)3 isoform displayed an increased abundance during muscle aging, which was independently verified by immunoblotting of differently aged human skeletal muscle samples. Since the CA3 isoform is relatively muscle-specific and exhibits a fibre type-specific expression pattern, this enzyme may represent an interesting new biomarker of sarcopenia. Increased levels of CA are indicative of an increased demand of CO₂-removal in senescent muscle, and also suggest age-related fibre type shifting to slower-contracting muscles during human aging.

Amyotrophic lateral sclerosis is the most common form of motor neuron disease in adult patients and characterized by progressive paralysis. The wobbler mouse (phenotype WR, genotype wr/wr) is an established animal model of human motor neuron disease and is characterized by a large variety of cellular abnormalities including muscular atrophy. In analogy to recent proteomic studies of cerebrospinal fluid and spinal cord, we have used here fluorescence difference in-gel electrophoresis to analyze global changes in the skeletal muscle proteome from WR versus normal mice. Relative concentrations of 21 proteins were found to be increased and 3 proteins were decreased. Mass spectrometric analysis identified these proteins to be associated with key metabolic pathways, the contractile apparatus, intermediate filaments and the cellular stress response. Drastically increased levels of the glycolytic enzyme glyceraldehyde- 3-phosphate dehydrogenase were confirmed by immunoblotting and this finding agrees with the idea of an oxidative-to-glycolytic shift in disease-related muscular atrophy. The establishment of novel disease-specific biomarkers of motor neuron disease might be helpful in the design of improved diagnostic tools and the identification of novel therapeutic targets.

National Institute for Cellular Biotechnology, Dublin City University, Dublin, IrelandInhumanglobozoospermia,round-headedspermatozoalackanacrosomeandthereforecannotproperlyinteractwithoocytes.Inthewobbler(WR)mouse,anL967Qmissensemutationinthevesicular protein-sorting factor VPS54 causes motor neuron degeneration and globozoosper-mia. Although electron microscopy of WR testis shows all major components of spermatoge-nesis, they appear in a deranged morphology that prevents the formation of the acrosome. Inordertodetermineproteome-widechanges,affectedtesteswereanalysedby2D-DIGEandMS.The concentration of 8 proteins was increased and that of 35 proteins decreased as comparedto wild type. Mass spectrometric analysis identified proteins with an altered concentration tobe associated with metabolite transport, fatty acid metabolism, cellular interactions, micro-tubule assembly and stress response (chaperones Hsp70–2 and Hsp90!). Minor changes wereobservedforproteinsinvolvedincellredoxhomeostasis,cytoskeletonformation,PTMs,detox-ification and metabolism. The most dramatically decreased protein in WR testis was identifiedas fatty acid binding protein FABP3, as confirmed by immunoblot analysis. We conclude thatamissensemutationinVPS54,anessentialcomponentoftheGolgi-associatedretrogradeprotein complex, not only prevents the formation of an acrosome but also initiates a cascade ofmetabolic abnormalities and a stress reaction.

Autoimmune diseases are systemic and organ–specific inflammatory conditions involving a cell-mediated immune response against self tissues. Whilst it is known that they are charac‐ terized by autoantibodies in both the systemic fluid and tissues [1, 2], the detailed aetiology and pathogenesis of auto-immune diseases are still poorly understood [3, 4]. These types of diseases occur when self molecules, often unknown antigens (auto-antigens), are seen by the immune system as non-self and are thereby attacked immunologically by the production of autoantibodies against them. In this process, the immune system mistakenly reacts with the body’s own cellular gears as if they were foreign antigens. The clinical presentations of autoimmune diseases vary with the disease course and delay in diagnosis as well as inappropri‐ ate therapy, increases tissue damage [3, 5].

Purpose Combined hormone and radiation therapy (CHRT) is one of the principle curative regimes for localised prostate cancer (PCa). Following treatment, many patients subsequently experience disease recurrence however; current diagnostics tests fail to predict the onset of disease recurrence. Biomarkers that address this issue would be of significant advantage. Experimental design Label-free LC-MS/MS for protein biomarker discovery and MRM for targeted confirmation were applied to patient serum samples accrued in a non-interventional clinical trial of CHRT. Results Analysis of time-matched patient samples from a patient with disease recurrence compared with a time match disease-free individual supported the identification of 287 proteins. Of these, 141 proteins were quantified, 95 proteins changed in their expression (P ≤ 0.05 and ≥1.5-fold change) and of these 16 were selected for MRM confirmation. The protein expression changes observed in the label-free LC-MS/MS and MRM analysis were found to be highly correlated (R2 = 0.85). Conclusions and clinical relevance The establishment of a clinical trial to support the acquisition of samples and development of a pipeline for MS-based biomarker discovery and validation should contribute to the identification of a serum protein signature to predict or monitor the outcome of treatment of patients with PCa.

Myotonia is a symptom of various genetic and acquired skeletal muscular disorders and is characterized by hyperexcitability of the sarcolemma. Here, we have performed a comparative proteomic study of the genetic mouse models ADR, MTO and MTO*5J of human congenital myotonia in order to determine myotonia-specific changes in the global protein complement of gastrocnemius muscle. Proteomic analyses of myotonia in the mouse, which is caused by mutations in the gene encoding the muscular chloride channel Clc1, revealed a generally perturbed protein expression pattern in severely affected ADR and MTO muscle, but less pronounced alterations in mildly diseased MTO*5J mice. Alterations were found in major metabolic pathways, the contractile machinery, ion handling elements, the cellular stress response and cell signaling mechanisms, clearly confirming a glycolytic-to-oxidative transformation process in myotonic fast muscle. In the long-term, a detailed biomarker signature of myotonia will improve our understanding of the pathobiochemical processes underlying this disorder and be helpful in determining how a single mutation in a tissue-specific gene can trigger severe downstream effects on the expression levels of a very large number of genes in contractile tissues.

The sarcoplasmic reticulum from skeletal muscle constitutes an elaborate membrane system that contains a considerable number of integral and very large proteins that exist in highly complex supramolecular clusters. Conventional proteomics using two-dimensional gel electrophoresis greatly underestimates the presence of these proteins. Here, we have applied one-dimensional gradient gels and on-membrane digestion to overcome this technical problem. Mass spectrometric analysis has determined the presence of 31 distinct protein species in the sarcoplasmic reticulum, including key Ca2+-handling proteins such as the ryanodine receptor, Ca2+-ATPase, calsequestrin and sarcalumenin. Immunoblotting confirmed the relative position of these Ca2+-regulatory elements in analytical gel replicas. Interestingly, aldolase and phosphofructokinase were found to be present in the purified sarcoplasmic reticulum, supporting the idea of a close physical coupling between the glycolytic pathway and the energy-dependent sarcoplasmic reticulum. Hence, on-membrane digestion is highly suitable as the method of choice for studying integral and high-molecular-mass proteins in proteomic studies.

Mitochondria are of central importance for energy generation in skeletal muscles. Expression changes or functional alterations in mitochondrial enzymes play a key role during myogenesis, fibre maturation, and various neuromuscular pathologies, as well as natural fibre aging. Mass spectrometry-based proteomics suggests itself as a convenient large-scale and high-throughput approach to catalogue the mitochondrial protein complement and determine global changes during health and disease. This paper gives a brief overview of the relatively new field of mitochondrial proteomics and discusses the findings from recent proteomic surveys of mitochondrial elements in aged skeletal muscles. Changes in the abundance, biochemical activity, subcellular localization, and/or posttranslational modifications in key mitochondrial enzymes might be useful as novel biomarkers of aging. In the long term, this may advance diagnostic procedures, improve the monitoring of disease progression, help in the testing of side effects due to new drug regimes, and enhance our molecular understanding of age-related muscle degeneration.

Contractile weakness and loss of muscle mass are critical features of the aging process in mammalians. Age-related fibre wasting has a profound effect on muscle metabolism, fibre type distribution and the overall physiological integrity of the neuromuscular system. This study has used mass spectrometry-based proteomics to investigate the fate of the aging rat muscle proteome. Using nonionic detergent phase extraction, this report shows that the aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in both the detergent-extracted fraction and the aqueous protein complement from senescent muscle tissue. In the detergent-extracted fraction, the expression of ATP synthase, isocitrate dehydrogenase, enolase, tropomyosin and beta-actin was increased. Different isoforms of creatine kinase and prohibitin showed differential changes. In the aqueous fraction, malate dehydrogenase, sulfotransferase, triosephosphate isomerase, aldolase, cofilin-2 and lactate dehydrogenase showed increased levels. Interestingly, differential effects on dissimilar 2-D spots of the same protein species were shown for Cu/Zn superoxide dismutase, albumin, annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The majority of altered proteins were found to be involved in mitochondrial metabolism, glycolysis, metabolic transportation, regulatory processes, the cellular stress response, detoxification mechanisms and muscle contraction.

Graphical abstract, Highlights ⿢ A systematic workflow for unbiased label-free LC⿿MS/MS analysis of laser capture microdissected material is presented. ⿢ This LCM proteomics workflow was applied to fresh frozen tissue sections from prostate cancer patients. ⿢ Expert annotation of regions of tumor for dissection from the sections of heterogenous tissue was undertaken by telepathology. ⿢ Requirements for sample loading to support reproducible semi-quantitative analysis of protein expression were determined. ⿢ To support tumor characterisation the LCM proteomics workflow was used to identify approximately 2000 proteins from tumor regions acquired from patient tissue sections., Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections. Here, we present an optimized workflow for coupling LCM to LC⿿MS/MS including: sectioning of tissue, a standard LCM workflow, protein digestion and advanced LC⿿MS/MS. Soluble proteins extracted from benign epithelial cells, their associated stroma, tumor epithelial cells and their associated stromal cells from a single patient tissue sample were digested and profiled using advanced LC⿿MS/MS. The correlation between technical replicates was R2 = 0.99 with a mean % CV of 9.55% ± 8.73. The correlation between sample replicates was R2 = 0.97 with a mean % CV of 13.83% ± 10.17. This represents a robust, systematic approach for profiling of the tumor microenvironment using LCM coupled to label-free LC⿿MS/MS.

One of the most important post-translational modifications is represented by phosphorylation on tyrosine, threonine and serine residues. Since abnormal phosphorylation is associated with various pathologies, it was of interest to perform a phosphoproteomic profiling of age-related skeletal muscle degeneration. We used the fluorescent phospho-specific Pro-Q Diamond dye to determine whether changes in the overall phosphorylation of the soluble skeletal muscle proteome differs significantly between young adult and senescent fibres. As an established model system of sarcopenia, we employed 30-month-old rat gastrocnemius fibres. Following the mass spectrometric identification of 59 major 2-D phosphoprotein landmark spots, the fluorescent dye staining survey revealed that 22 muscle proteins showed a differential expression pattern between 3-month- and 30-month-old muscle. Increased phosphorylation levels were shown for myosin light chain 2, tropomyosin alpha, lactate dehydrogenase, desmin, actin, albumin and aconitase. In contrast, decreased phospho-specific dye binding was observed for cytochrome c oxidase, creatine kinase and enolase. Thus, aging-induced alterations in phosphoproteins appear to involve the contractile machinery and the cytoskeleton, as well as the cytosolic and mitochondrial metabolism. This confirms that sarcopenia of old age is a complex neuromuscular pathology that is associated with drastic changes in the abundance and structure of key muscle proteins.