Proteomes

20 pages, 5257 KiB

Open AccessArticle

A Proteomic View of Cellular Responses to Anticancer Quinoline-Copper Complexes

by Bastien Dalzon, Joanna Bons, Hélène Diemer, Véronique Collin-Faure, Caroline Marie-Desvergne, Muriel Dubosson, Sarah Cianferani, Christine Carapito and Thierry Rabilloud

Proteomes 2019, 7(2), 26; https://doi.org/10.3390/proteomes7020026 - 24 Jun 2019

Cited by 12 | Viewed by 4364

Abstract

Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a [...] Read more.

Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a classical proteomic approach based on two-dimensional elelctrophoresis to investigate the mechanisms of action of a hydroxyquinoline-copper complex, which shows promising anticancer activities, using the leukemic cell line RAW264.7 as the biological target. Pathway analysis of the modulated proteins highlighted changes in the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, and carbon metabolism or oxido-reduction. In line with these prteomic-derived hypotheses, targeted validation experiments showed that the hydroxyquinoline-copper complex induces a massive reduction in free glutathione and a strong alteration in the actin cytoskeleton, suggesting a multi-target action of the hydroxyquinoline-copper complex on cancer cells. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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Graphical abstract

23 pages, 7219 KiB

Open AccessReview

Characterization of Contractile Proteins from Skeletal Muscle Using Gel-Based Top-Down Proteomics

by Paul Dowling, Margit Zweyer, Dieter Swandulla and Kay Ohlendieck

Proteomes 2019, 7(2), 25; https://doi.org/10.3390/proteomes7020025 - 20 Jun 2019

Cited by 23 | Viewed by 8952

Abstract

The mass spectrometric analysis of skeletal muscle proteins has used both peptide-centric and protein-focused approaches. The term ‘top-down proteomics’ is often used in relation to studying purified proteoforms and their post-translational modifications. Two-dimensional gel electrophoresis, in combination with peptide generation for the identification [...] Read more.

The mass spectrometric analysis of skeletal muscle proteins has used both peptide-centric and protein-focused approaches. The term ‘top-down proteomics’ is often used in relation to studying purified proteoforms and their post-translational modifications. Two-dimensional gel electrophoresis, in combination with peptide generation for the identification and characterization of intact proteoforms being present in two-dimensional spots, plays a critical role in specific applications of top-down proteomics. A decisive bioanalytical advantage of gel-based and top-down approaches is the initial bioanalytical focus on intact proteins, which usually enables the swift identification and detailed characterisation of specific proteoforms. In this review, we describe the usage of two-dimensional gel electrophoretic top-down proteomics and related approaches for the systematic analysis of key components of the contractile apparatus, with a special focus on myosin heavy and light chains and their associated regulatory proteins. The detailed biochemical analysis of proteins belonging to the thick and thin skeletal muscle filaments has decisively improved our biochemical understanding of structure-function relationships within the contractile apparatus. Gel-based and top-down proteomics has clearly established a variety of slow and fast isoforms of myosin, troponin and tropomyosin as excellent markers of fibre type specification and dynamic muscle transition processes. Full article

(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)

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9 pages, 197 KiB

Open AccessEditorial

Editorial for Special Issue: Neuroproteomics

by Kenneth R. Williams and Angus C. Nairn

Proteomes 2019, 7(2), 24; https://doi.org/10.3390/proteomes7020024 - 31 May 2019

Cited by 1 | Viewed by 3783

Abstract

Recent advances in mass spectrometry (MS) instrumentation [...] Full article

(This article belongs to the Special Issue Neuroproteomics)

10 pages, 693 KiB

Open AccessFeature PaperReview

Different Types of Cellular Stress Affect the Proteome Composition of Small Extracellular Vesicles: A Mini Review

by Agata Abramowicz, Piotr Widłak and Monika Pietrowska

Proteomes 2019, 7(2), 23; https://doi.org/10.3390/proteomes7020023 - 23 May 2019

Cited by 29 | Viewed by 5133

Abstract

Extracellular vesicles (EVs) are well-known mediators of the cellular response to different stress factors, yet the exact mechanism of their action remains unclear. Hence, the characterization of their cargo, consisting of proteins, nucleic acids, and different classes of metabolites, helps to elucidate an [...] Read more.

Extracellular vesicles (EVs) are well-known mediators of the cellular response to different stress factors, yet the exact mechanism of their action remains unclear. Hence, the characterization of their cargo, consisting of proteins, nucleic acids, and different classes of metabolites, helps to elucidate an understanding of their function in stress-related communication. The unexpected diversity and complexity of these vesicles requires the incorporation of multiple technologically advanced approaches in EV-oriented studies. This mini review focuses on the invaluable role of proteomics, especially mass spectrometry-based tools, in the investigation of the role of small EVs in their response to stress. Though relatively few experimental works address this issue to date, the available data indicate that stress conditions would affect the composition of protein cargo of vesicles released by stressed cells, as evidenced by the functional importance of such changes in the context of the response of recipient cells. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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12 pages, 253 KiB

Open AccessReview

Challenges in the Isolation and Proteomic Analysis of Cancer Exosomes—Implications for Translational Research

by Jadwiga Jablonska, Monika Pietrowska, Sonja Ludwig, Stephan Lang and Basant Kumar Thakur

Proteomes 2019, 7(2), 22; https://doi.org/10.3390/proteomes7020022 - 15 May 2019

Cited by 21 | Viewed by 5601

Abstract

Exosomes belong to the group of extracellular vesicles (EVs) that derive from various cell populations and mediate intercellular communication in health and disease. Like hormones or cytokines, exosomes released by cells can play a potent role in the communication between the cell of [...] Read more.

Exosomes belong to the group of extracellular vesicles (EVs) that derive from various cell populations and mediate intercellular communication in health and disease. Like hormones or cytokines, exosomes released by cells can play a potent role in the communication between the cell of origin and distant cells in the body to maintain homeostatic or pathological processes, including tumorigenesis. The nucleic acids, and lipid and protein cargo present in the exosomes are involved in a myriad of carcinogenic processes, including cell proliferation, tumor angiogenesis, immunomodulation, and metastasis formation. The ability of exosomal proteins to mediate direct functions by interaction with other cells qualifies them as tumor-specific biomarkers and targeted therapeutic approaches. However, the heterogeneity of plasma-derived exosomes consistent of (a) exosomes derived from all kinds of body cells, including cancer cells and (b) contamination of exosome preparation with other extracellular vesicles, such as apoptotic bodies, makes it challenging to obtain solid proteomics data for downstream clinical application. In this manuscript, we review these challenges beginning with the choice of different isolation methods, through the evaluation of obtained exosomes and limitations in the process of proteome analysis of cancer-derived exosomes to identify novel protein targets with functional impact in the context of translational oncology. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

16 pages, 744 KiB

Open AccessReview

Melanoma-Derived Extracellular Vesicles: Focus on Their Proteome

by Magdalena Surman, Ewa Stępień and Małgorzata Przybyło

Proteomes 2019, 7(2), 21; https://doi.org/10.3390/proteomes7020021 - 13 May 2019

Cited by 21 | Viewed by 6133

Abstract

Malignant melanoma is one of the most aggressive types of cancer, and its incidence is increasing rapidly each year. Despite the extensive research into improved diagnostic and treatment methods, early detection and disease constraint still present significant challenges. As successful isolation protocols have [...] Read more.

Malignant melanoma is one of the most aggressive types of cancer, and its incidence is increasing rapidly each year. Despite the extensive research into improved diagnostic and treatment methods, early detection and disease constraint still present significant challenges. As successful isolation protocols have been developed, extracellular vesicles (EVs) have become the subject of extensive investigation in terms of their role in cancer progression and as a possible source of disease biomarkers. Besides functional studies, quantitative and qualitative proteomics have recently emerged as promising tools for the advancement of melanoma biomarkers. Nevertheless, the amount of data concerning the proteome of melanoma-derived EVs is still very limited. In this review we cover the current knowledge on protein content of melanoma-derived EVs, with a focus on their potential role in the development and progression of melanomas. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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17 pages, 2825 KiB

Open AccessFeature PaperArticle

Comparison of Proteome Composition of Serum Enriched in Extracellular Vesicles Isolated from Polycythemia Vera Patients and Healthy Controls

by Anna Fel, Aleksandra E. Lewandowska, Petro E. Petrides and Jacek R. Wiśniewski

Proteomes 2019, 7(2), 20; https://doi.org/10.3390/proteomes7020020 - 06 May 2019

Cited by 21 | Viewed by 5495

Abstract

Extracellular vesicles (EVs), e.g., exosomes and microvesicles, are one of the main networks of intercellular communication. In myeloproliferative neoplasms, such as polycythemia vera (PV), excess of EVs originating from overabundant blood cells can directly contribute to thrombosis through their procoagulant activity. However, the [...] Read more.

Extracellular vesicles (EVs), e.g., exosomes and microvesicles, are one of the main networks of intercellular communication. In myeloproliferative neoplasms, such as polycythemia vera (PV), excess of EVs originating from overabundant blood cells can directly contribute to thrombosis through their procoagulant activity. However, the proteomic composition of these vesicles in PV patients has not been investigated before. In this work, we examined the proteomic composition of serum EVs of PV patients in comparison to healthy controls. We processed EV-enriched serum samples using the Multiple Enzyme Filter Aided Sample Preparation approach (MED-FASP), conducted LC-MS/MS measurements on a Q-Exactive HF-X mass spectrometer, and quantitatively analyzed the absolute concentrations of identified proteins by the Total Protein Approach (TPA). Thirty-eight proteins were present at statistically significant different concentrations between PV patients’ study group and healthy controls’ group. The main protein components deregulated in PV were primarily related to excessive amounts of cells, increased platelet activation, elevated immune and inflammatory response, and high concentrations of procoagulant and angiogenic agents. Our study provides the first quantitative analysis of the serum EVs’ proteome in PV patients. This new knowledge may contribute to a better understanding of the secondary systemic effects of PV disease and further development of diagnostic or therapeutic procedures. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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11 pages, 3145 KiB

Open AccessArticle

A Preliminary Metagenome Analysis Based on a Combination of Protein Domains

by Yoji Igarashi, Daisuke Mori, Susumu Mitsuyama, Kazutoshi Yoshitake, Hiroaki Ono, Tsuyoshi Watanabe, Yukiko Taniuchi, Tomoko Sakami, Akira Kuwata, Takanori Kobayashi, Yoshizumi Ishino, Shugo Watabe, Takashi Gojobori and Shuichi Asakawa

Proteomes 2019, 7(2), 19; https://doi.org/10.3390/proteomes7020019 - 29 Apr 2019

Viewed by 3764

Abstract

Metagenomic data have mainly been addressed by showing the composition of organisms based on a small part of a well-examined genomic sequence, such as ribosomal RNA genes and mitochondrial DNAs. On the contrary, whole metagenomic data obtained by the shotgun sequence method have [...] Read more.

Metagenomic data have mainly been addressed by showing the composition of organisms based on a small part of a well-examined genomic sequence, such as ribosomal RNA genes and mitochondrial DNAs. On the contrary, whole metagenomic data obtained by the shotgun sequence method have not often been fully analyzed through a homology search because the genomic data in databases for living organisms on earth are insufficient. In order to complement the results obtained through homology-search-based methods with shotgun metagenomes data, we focused on the composition of protein domains deduced from the sequences of genomes and metagenomes, and we utilized them in characterizing genomes and metagenomes, respectively. First, we compared the relationships based on similarities in the protein domain composition with the relationships based on sequence similarities. We searched for protein domains of 325 bacterial species produced using the Pfam database. Next, the correlation coefficients of protein domain compositions between every pair of bacteria were examined. Every pairwise genetic distance was also calculated from 16S rRNA or DNA gyrase subunit B. We compared the results of these methods and found a moderate correlation between them. Essentially, the same results were obtained when we used partial random 100 bp DNA sequences of the bacterial genomes, which simulated raw sequence data obtained from short-read next-generation sequences. Then, we applied the method for analyzing the actual environmental data obtained by shotgun sequencing. We found that the transition of the microbial phase occurred because the seasonal change in water temperature was shown by the method. These results showed the usability of the method in characterizing metagenomic data based on protein domain compositions. Full article

(This article belongs to the Special Issue Microbial Proteomics II)

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12 pages, 1152 KiB

Open AccessFeature PaperArticle

Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components

by Mateusz Smolarz, Monika Pietrowska, Natalia Matysiak, Łukasz Mielańczyk and Piotr Widłak

Proteomes 2019, 7(2), 18; https://doi.org/10.3390/proteomes7020018 - 28 Apr 2019

Cited by 67 | Viewed by 8817

Abstract

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from [...] Read more.

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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14 pages, 4422 KiB

Open AccessArticle

Isolation and Characterization of Small Extracellular Vesicles from Porcine Blood Plasma, Cerebrospinal Fluid, and Seminal Plasma

by Helena Kupcova Skalnikova, Bozena Bohuslavova, Karolina Turnovcova, Jana Juhasova, Stefan Juhas, Marie Rodinova and Petr Vodicka

Proteomes 2019, 7(2), 17; https://doi.org/10.3390/proteomes7020017 - 25 Apr 2019

Cited by 31 | Viewed by 8848

Abstract

Extracellular vesicles (EVs) are a highly attractive subject of biomedical research as possible carriers of nucleic acid and protein biomarkers. EVs released to body fluids enable indirect access to inner organs by so-called “liquid biopsies”. Obtaining a high-quality EV sample with minimum contaminants [...] Read more.

Extracellular vesicles (EVs) are a highly attractive subject of biomedical research as possible carriers of nucleic acid and protein biomarkers. EVs released to body fluids enable indirect access to inner organs by so-called “liquid biopsies”. Obtaining a high-quality EV sample with minimum contaminants is crucial for proteomic analyses using LC–MS/MS or other techniques. However, the EV content in various body fluids largely differs, which may hamper subsequent analyses. Here, we present a comparison of extracellular vesicle yields from blood plasma, cerebrospinal fluid, and seminal plasma using an experimental pig model. Pigs are widely used in biomedical research as large animal models with anatomy and physiology close to those of humans and enable studies (e.g., of the nervous system) that are unfeasible in humans. EVs were isolated from body fluids by differential centrifugation followed by ultracentrifugation. EVs were characterized according to protein yields and to the quality of the isolated vesicles (e.g., size distribution, morphology, positivity for exosome markers). In our experimental setting, substantial differences in EV amounts were identified among body fluids, with the seminal plasma being the richest EV source. The yields of pellet proteins from ultracentrifugation of 1 mL of porcine body fluids may help to estimate body fluid input volumes to obtain sufficient samples for subsequent proteomic analyses. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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11 pages, 2426 KiB

Open AccessArticle

The Proteome of Tetrasphaera elongata is adapted to Changing Conditions in Wastewater Treatment Plants

by Florian-Alexander Herbst, Morten S. Dueholm, Reinhard Wimmer and Per Halkjær Nielsen

Proteomes 2019, 7(2), 16; https://doi.org/10.3390/proteomes7020016 - 25 Apr 2019

Cited by 18 | Viewed by 5512

Abstract

The activated sludge in wastewater treatment plants (WWTP) designed for enhanced biological phosphorus removal (EBPR) experiences periodically changing nutrient and oxygen availability. Tetrasphaera is the most abundant genus in Danish WWTP and represents up to 20–30% of the activated sludge community based on [...] Read more.

The activated sludge in wastewater treatment plants (WWTP) designed for enhanced biological phosphorus removal (EBPR) experiences periodically changing nutrient and oxygen availability. Tetrasphaera is the most abundant genus in Danish WWTP and represents up to 20–30% of the activated sludge community based on 16S rRNA amplicon sequencing and quantitative fluorescence in situ hybridization analyses, although the genus is in low abundance in the influent wastewater. Here we investigated how Tetrasphaera can successfully out-compete most other microorganisms in such highly dynamic ecosystems. To achieve this, we analyzed the physiological adaptations of the WWTP isolate T. elongata str. LP2 during an aerobic to anoxic shift by label-free quantitative proteomics and NMR-metabolomics. Escherichia coli was used as reference organism as it shares several metabolic capabilities and is regularly introduced to wastewater treatment plants without succeeding there. When compared to E. coli, only minor changes in the proteome of T. elongata were observed after the switch to anoxic conditions. This indicates that metabolic pathways for anaerobic energy harvest were already expressed during the aerobic growth. This allows continuous growth of Tetrasphaera immediately after the switch to anoxic conditions. Metabolomics furthermore revealed that the substrates provided were exploited far more efficiently by Tetrasphaera than by E. coli. These results suggest that T. elongata prospers in the dynamic WWTP environment due to adaptation to the changing environmental conditions. Full article

(This article belongs to the Special Issue Microbial Proteomics II)

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11 pages, 1518 KiB

Open AccessArticle

More than a Toxin: Protein Inventory of Clostridium tetani Toxoid Vaccines

by Jens Möller, Max Edmund Kraner and Andreas Burkovski

Proteomes 2019, 7(2), 15; https://doi.org/10.3390/proteomes7020015 - 16 Apr 2019

Cited by 14 | Viewed by 6991

Abstract

Clostridium tetani is the etiological agent of tetanus, a life-threatening bacterial infection. The most efficient protection strategy against tetanus is a vaccination with the C. tetani neurotoxin, which is inactivated by formaldehyde-crosslinking. Since we assumed that besides the tetanus toxin, other proteins of [...] Read more.

Clostridium tetani is the etiological agent of tetanus, a life-threatening bacterial infection. The most efficient protection strategy against tetanus is a vaccination with the C. tetani neurotoxin, which is inactivated by formaldehyde-crosslinking. Since we assumed that besides the tetanus toxin, other proteins of C. tetani may also be present in toxoid preparations, we analyzed commercially available vaccines from different countries in respect to their protein content using mass spectrometry. In total 991 proteins could be identified in all five analyzed vaccines, 206 proteins were common in all analyzed vaccines and 54 proteins from the 206 proteins were potential antigens. The additionally present proteins may contribute at least partially to protection against C. tetani infection by supporting the function of the vaccine against the devastating effects of the tetanus toxin indirectly. Two different label-free protein quantification methods were applied for an estimation of protein contents. Similar results were obtained with a Total Protein Approach (TPA)-based method and Protein Discoverer 2.2 software package based on the minora algorithm. Depending on the tetanus toxoid vaccine and the quantification method used, tetanus neurotoxin contributes between 14 and 76 % to the total C. tetani protein content and varying numbers of other C. tetani proteins were detected. Full article

(This article belongs to the Special Issue Microbial Proteomics II)

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13 pages, 1918 KiB

Open AccessCommunication

Extracellular Vesicle Integrins Distinguish Unique Cancers

by Stephanie N. Hurwitz and David G. Meckes, Jr.

Proteomes 2019, 7(2), 14; https://doi.org/10.3390/proteomes7020014 - 11 Apr 2019

Cited by 45 | Viewed by 5948

Abstract

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote [...] Read more.

The proteomic profile of extracellular vesicles (EVs) has been of increasing interest, particularly in understanding cancer growth, drug resistance, and metastatic behavior. Emerging data suggest that cancer-derived EVs carry an array of oncogenic cargo, including certain integrin proteins that may, in turn, promote cell detachment, migration, and selection of future metastatic sites. We previously reported a large comparison of secreted vesicle protein cargo across sixty diverse human cancer cell lines. Here, we analyze the distinct integrin profiles of these cancer EVs. We further demonstrate the enrichment of integrin receptors in cancer EVs compared to vesicles secreted from benign epithelial cells. The total EV integrin levels, including the quantity of integrins α6, αv, and β1 correlate with tumor stage across a variety of epithelial cancer cells. In particular, integrin α6 also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumors. This study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumor stage. Differential expression of integrins across cancer cells and selective packaging of integrins into EVs may contribute to further understanding the development and progression of tumor growth and metastasis across a variety of cancer types. Full article

(This article belongs to the Special Issue Proteomes of Extracellular Vesicles)

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15 pages, 2001 KiB

Open AccessArticle

Calreticulin as A Novel Potential Metastasis-Associated Protein in Myxoid Liposarcoma, as Revealed by Two-Dimensional Difference Gel Electrophoresis

by Takashi Tajima, Fusako Kito, Akihiko Yoshida, Akira Kawai and Tadashi Kondo

Proteomes 2019, 7(2), 13; https://doi.org/10.3390/proteomes7020013 - 10 Apr 2019

Cited by 3 | Viewed by 3742

Abstract

Myxoid liposarcoma (MLS) is a mesenchymal malignancy. To identify innovate seeds for clinical applications, we examined the proteomes of primary tumor tissues from 10 patients with MLS with different statuses of postoperative metastasis. The protein expression profiles of tumor tissues were created, and [...] Read more.

Myxoid liposarcoma (MLS) is a mesenchymal malignancy. To identify innovate seeds for clinical applications, we examined the proteomes of primary tumor tissues from 10 patients with MLS with different statuses of postoperative metastasis. The protein expression profiles of tumor tissues were created, and proteins with differential expression associated with postoperative metastasis were identified by two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. The validation was performed using specific antibodies and in vitro analyses. Using 2D-DIGE, we observed 1726 protein species and identified proteins with unique expression levels in metastatic MLS. We focused on the overexpression of calreticulin in metastatic MLS. The higher expression of calreticulin was confirmed by Western blotting, and gene silencing assays demonstrated that reduced expression of calreticulin inhibited cell growth and invasion. Our findings suggested the important roles of calreticulin in MLS metastasis and supported its potential utility as a prognostic biomarker in MLS. Further investigations of the functional properties of calreticulin and other proteins identified in this study will improve our understanding of the biology of MLS and facilitate novel clinical applications. Full article

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22 pages, 3348 KiB

Open AccessArticle

Development of Targeted Mass Spectrometry-Based Approaches for Quantitation of Proteins Enriched in the Postsynaptic Density (PSD)

by Rashaun S. Wilson, Navin Rauniyar, Fumika Sakaue, TuKiet T. Lam, Kenneth R. Williams and Angus C. Nairn

Proteomes 2019, 7(2), 12; https://doi.org/10.3390/proteomes7020012 - 02 Apr 2019

Cited by 15 | Viewed by 7161

Abstract

The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in [...] Read more.

The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli, and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. Here, we report on the development of two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards and identified and quantified over 2100 proteins through a pre-determined spectral library using a data-independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue. Full article

(This article belongs to the Special Issue Neuroproteomics)

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13 pages, 932 KiB

Open AccessTechnical Note

Terminomics Methodologies and the Completeness of Reductive Dimethylation: A Meta-Analysis of Publicly Available Datasets

by Mariella Hurtado Silva, Iain J. Berry, Natalie Strange, Steven P. Djordjevic and Matthew P. Padula

Proteomes 2019, 7(2), 11; https://doi.org/10.3390/proteomes7020011 - 29 Mar 2019

Cited by 3 | Viewed by 3934

Abstract

Methods for analyzing the terminal sequences of proteins have been refined over the previous decade; however, few studies have evaluated the quality of the data that have been produced from those methodologies. While performing global N-terminal labelling on bacteria, we observed that the [...] Read more.

Methods for analyzing the terminal sequences of proteins have been refined over the previous decade; however, few studies have evaluated the quality of the data that have been produced from those methodologies. While performing global N-terminal labelling on bacteria, we observed that the labelling was not complete and investigated whether this was a common occurrence. We assessed the completeness of labelling in a selection of existing, publicly available N-terminomics datasets and empirically determined that amine-based labelling chemistry does not achieve complete labelling and potentially has issues with labelling amine groups at sequence-specific residues. This finding led us to conduct a thorough review of the historical literature that showed that this is not an unexpected finding, with numerous publications reporting incomplete labelling. These findings have implications for the quantitation of N-terminal peptides and the biological interpretations of these data. Full article

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Proteomes, Volume 7, Issue 2 (June 2019) – 16 articles

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