Proteomes

18 pages, 2760 KiB

Open AccessArticle

Data-Independent Acquisition Enables Robust Quantification of 400 Proteins in Non-Depleted Canine Plasma

by Halley Gora Ravuri, Zainab Noor, Paul C. Mills, Nana Satake and Pawel Sadowski

Proteomes 2022, 10(1), 9; https://doi.org/10.3390/proteomes10010009 - 28 Feb 2022

Cited by 3 | Viewed by 4077

Mass spectrometry-based plasma proteomics offers a major advance for biomarker discovery in the veterinary field, which has traditionally been limited to quantification of a small number of proteins using biochemical assays. The development of foundational data and tools related to sequential window acquisition [...] Read more.

Mass spectrometry-based plasma proteomics offers a major advance for biomarker discovery in the veterinary field, which has traditionally been limited to quantification of a small number of proteins using biochemical assays. The development of foundational data and tools related to sequential window acquisition of all theoretical mass spectra (SWATH)-mass spectrometry has allowed for quantitative profiling of a significant number of plasma proteins in humans and several animal species. Enabling SWATH in dogs enhances human biomedical research as a model species, and significantly improves diagnostic and disease monitoring capability. In this study, a comprehensive peptide spectral library specific to canine plasma proteome was developed and evaluated using SWATH for protein quantification in non-depleted dog plasma. Specifically, plasma samples were subjected to various orthogonal fractionation and digestion techniques, and peptide fragmentation data corresponding to over 420 proteins was collected. Subsequently, a SWATH-based assay was introduced that leveraged the developed resource and that enabled reproducible quantification of 400 proteins in non-depleted plasma samples corresponding to various disease conditions. The ability to profile the abundance of such a significant number of plasma proteins using a single method in dogs has the potential to accelerate biomarker discovery studies in this species. Full article

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17 pages, 1952 KiB

Open AccessArticle

Molecular Dynamics Study of Citrullinated Proteins Associated with the Development of Rheumatoid Arthritis

by Amir Taldaev, Vladimir Rudnev, Liudmila Kulikova, Kirill Nikolsky, Alexander Efimov, Kristina Malsagova and Anna Kaysheva

Proteomes 2022, 10(1), 8; https://doi.org/10.3390/proteomes10010008 - 11 Feb 2022

Cited by 4 | Viewed by 3369

Abstract

Biological activity regulation by protein post-translational modification (PTM) is critical for cell function, development, differentiation, and survival. Dysregulation of PTM proteins is present in various pathological conditions, including rheumatoid arthritis (RA). RA is a systemic autoimmune disease that primarily affects joints, and there [...] Read more.

Biological activity regulation by protein post-translational modification (PTM) is critical for cell function, development, differentiation, and survival. Dysregulation of PTM proteins is present in various pathological conditions, including rheumatoid arthritis (RA). RA is a systemic autoimmune disease that primarily affects joints, and there are three main types of protein PTMs associated with the development of this disease, namely, glycosylation, citrullination, and carbamylation. Glycosylation is important for the processing and presentation of antigen fragments on the cell surface and can modulate immunoglobulin activity. The citrullination of autoantigens is closely associated with RA, as evidenced by the presence of antibodies specific to citrullinated proteins in the serum of patients. Carbamylation and dysregulation have recently been associated with RA development in humans.In this study, we performed an overview analysis of proteins with post-translational modifications associated with the development of RA adverted in peer-reviewed scientific papers for the past 20 years. As a result of the search, a list of target proteins and corresponding amino acid sequences with PTM in RA was formed. Structural characteristics of the listed modified proteins were extracted from the Protein Data Bank. Then, molecular dynamics experiments of intact protein structures and corresponding structures with PTMs were performed regarding structures in the list announced in the ProtDB service. This study aimed to conduct a molecular dynamics study of intact proteins and proteins, including post-translational modification and protein citrullination, likely associated with RA development. We observed another exhibition of the fundamental physics concept, symmetry, at the submolecular level, unveiled as the autonomous repetitions of outside the protein structural motif performance globule corresponding to those in the whole protein molecule. Full article

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21 pages, 3029 KiB

Open AccessArticle

Bovine Peripheral Blood Derived Lymphocyte Proteome and Secretome Show Divergent Reaction of Bovine Immune Phenotypes after Stimulation with Pokeweed Mitogen

by Kristina J. H. Kleinwort, Roxane L. Degroote, Sieglinde Hirmer, Lucia Korbonits, Lea Lorenz, Armin M. Scholz, Stefanie M. Hauck and Cornelia A. Deeg

Proteomes 2022, 10(1), 7; https://doi.org/10.3390/proteomes10010007 - 08 Feb 2022

Cited by 3 | Viewed by 3182

Abstract

We recently identified a deviant bovine immune phenotype characterized by hyperproliferation of lymphocytes after polyclonal stimulation. This phenotype was first discovered in dams that responded to PregSure BVD vaccination by producing pathological antibodies, triggering the fatal disease “bovine neonatal pancytopenia” in calves. The [...] Read more.

We recently identified a deviant bovine immune phenotype characterized by hyperproliferation of lymphocytes after polyclonal stimulation. This phenotype was first discovered in dams that responded to PregSure BVD vaccination by producing pathological antibodies, triggering the fatal disease “bovine neonatal pancytopenia” in calves. The aim of the study was to gain deeper insights into molecular processes occurring in lymphocytes of immune phenotypes and the effect on their secretome after immune stimulation. Two discovery proteomic experiments were performed with unstimulated and Pokeweed Mitogen (PWM) stimulated lymphocytes, using label-free LC-MS/MS. In lymphocytes, 2447 proteins were quantified, and 1204 proteins were quantified in the secretome. Quantitative proteome analysis of immune deviant and control samples after PWM stimulation revealed clear differences. The increase in abundance of IL17A, IL17F, IL8, CCL5, LRRC59, and CLIC4 was higher in controls through mitogenic stimulation. In contrast, the abundance of IFNγ, IL2, IL2RA, CD83, and CD200 increased significantly more in immune deviant lymphocytes. Additional pathway enrichment analysis of differentially secreted proteins also yielded fundamental differences between the immune phenotypes. Our study provides a comprehensive dataset, which gives novel insights into proteome changes of lymphocytes from different bovine immune phenotypes. These differences point to the development of diverse immune responses of bovine immune phenotypes after immune stimulation. Full article

(This article belongs to the Section Animal Proteomics)

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2 pages, 162 KiB

Open AccessEditorial

Acknowledgment to Reviewers of Proteomes in 2021

by Proteomes Editorial Office

Proteomes 2022, 10(1), 6; https://doi.org/10.3390/proteomes10010006 - 25 Jan 2022

Viewed by 2174

Abstract

Rigorous peer-reviews are the basis of high-quality academic publishing [...] Full article

14 pages, 4504 KiB

Open AccessReview

Shotgun Proteomics as a Powerful Tool for the Study of the Proteomes of Plants, Their Pathogens, and Plant–Pathogen Interactions

by Sadegh Balotf, Richard Wilson, Robert S. Tegg, David S. Nichols and Calum R. Wilson

Proteomes 2022, 10(1), 5; https://doi.org/10.3390/proteomes10010005 - 19 Jan 2022

Cited by 16 | Viewed by 4914

Abstract

The interaction between plants and pathogenic microorganisms is a multifaceted process mediated by both plant- and pathogen-derived molecules, including proteins, metabolites, and lipids. Large-scale proteome analysis can quantify the dynamics of proteins, biological pathways, and posttranslational modifications (PTMs) involved in the plant–pathogen interaction. [...] Read more.

The interaction between plants and pathogenic microorganisms is a multifaceted process mediated by both plant- and pathogen-derived molecules, including proteins, metabolites, and lipids. Large-scale proteome analysis can quantify the dynamics of proteins, biological pathways, and posttranslational modifications (PTMs) involved in the plant–pathogen interaction. Mass spectrometry (MS)-based proteomics has become the preferred method for characterizing proteins at the proteome and sub-proteome (e.g., the phosphoproteome) levels. MS-based proteomics can reveal changes in the quantitative state of a proteome and provide a foundation for understanding the mechanisms involved in plant–pathogen interactions. This review is intended as a primer for biologists that may be unfamiliar with the diverse range of methodology for MS-based shotgun proteomics, with a focus on techniques that have been used to investigate plant–pathogen interactions. We provide a summary of the essential steps required for shotgun proteomic studies of plants, pathogens and plant–pathogen interactions, including methods for protein digestion, identification, separation, and quantification. Finally, we discuss how protein PTMs may directly participate in the interaction between a pathogen and its host plant. Full article

(This article belongs to the Section Plant Proteomics)

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15 pages, 1604 KiB

Open AccessArticle

Potential Tear Biomarkers for the Diagnosis of Parkinson’s Disease—A Pilot Study

by Arantxa Acera, Juan Carlos Gómez-Esteban, Ane Murueta-Goyena, Marta Galdos, Mikel Azkargorta, Felix Elortza, Noelia Ruzafa, Oliver Ibarrondo, Xandra Pereiro and Elena Vecino

Proteomes 2022, 10(1), 4; https://doi.org/10.3390/proteomes10010004 - 13 Jan 2022

Cited by 8 | Viewed by 5184

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. In this study, the tear proteome profile of patients with idiopathic PD (iPD, n = 24), carriers of the E46K-SNCA mutation (n = 3) and healthy control (CT, n [...] Read more.

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. In this study, the tear proteome profile of patients with idiopathic PD (iPD, n = 24), carriers of the E46K-SNCA mutation (n = 3) and healthy control (CT, n = 27) subjects was analyzed to identify candidate biomarkers for the diagnosis of PD. An observational, prospective and case-control pilot study was carried out, analyzing the participants tear samples by nano-liquid chromatography–mass spectrometry (nLC–MS/MS) and assessing their neurological impairment. The proteomic data obtained are available at ProteomeXchange with identifier 10.6019/PXD028811. These analyses led to the identification of 560 tear proteins, some of which were deregulated in PD patients and that have been implicated in immune responses, inflammation, apoptosis, collagen degradation, protein synthesis, defense, lipid transport and altered lysosomal function. Of these proteins, six were related to neurodegenerative processes and showed a good capacity to classify patients and controls. These findings revealed that certain proteins were upregulated in the tears of PD patients, mainly proteins involved in lysosomal function. Thus, in this study, tear proteins were identified that are implicated in neurodegeneration and that may be related to an aggressive disease phenotype in PD patients. Full article

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10 pages, 1321 KiB

Open AccessArticle

Standard Flow Multiplexed Proteomics (SFloMPro)—An Accessible Alternative to NanoFlow Based Shotgun Proteomics

by Benjamin C. Orsburn, Sierra D. Miller and Conor J. Jenkins

Proteomes 2022, 10(1), 3; https://doi.org/10.3390/proteomes10010003 - 13 Jan 2022

Cited by 4 | Viewed by 3188

Abstract

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the [...] Read more.

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 h. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 µg of labeled peptides per fraction with SFloMPro, compared to 1 µg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample. Full article

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13 pages, 1396 KiB

Open AccessArticle

Comparison of Different Label-Free Techniques for the Semi-Absolute Quantification of Protein Abundance

by Aarón Millán-Oropeza, Mélisande Blein-Nicolas, Véronique Monnet, Michel Zivy and Céline Henry

Proteomes 2022, 10(1), 2; https://doi.org/10.3390/proteomes10010002 - 07 Jan 2022

Cited by 10 | Viewed by 3818

Abstract

In proteomics, it is essential to quantify proteins in absolute terms if we wish to compare results among studies and integrate high-throughput biological data into genome-scale metabolic models. While labeling target peptides with stable isotopes allow protein abundance to be accurately quantified, the [...] Read more.

In proteomics, it is essential to quantify proteins in absolute terms if we wish to compare results among studies and integrate high-throughput biological data into genome-scale metabolic models. While labeling target peptides with stable isotopes allow protein abundance to be accurately quantified, the utility of this technique is constrained by the low number of quantifiable proteins that it yields. Recently, label-free shotgun proteomics has become the “gold standard” for carrying out global assessments of biological samples containing thousands of proteins. However, this tool must be further improved if we wish to accurately quantify absolute levels of proteins. Here, we used different label-free quantification techniques to estimate absolute protein abundance in the model yeast Saccharomyces cerevisiae. More specifically, we evaluated the performance of seven different quantification methods, based either on spectral counting (SC) or extracted-ion chromatogram (XIC), which were applied to samples from five different proteome backgrounds. We also compared the accuracy and reproducibility of two strategies for transforming relative abundance into absolute abundance: a UPS2-based strategy and the total protein approach (TPA). This study mentions technical challenges related to UPS2 use and proposes ways of addressing them, including utilizing a smaller, more highly optimized amount of UPS2. Overall, three SC-based methods (PAI, SAF, and NSAF) yielded the best results because they struck a good balance between experimental performance and protein quantification. Full article

(This article belongs to the Special Issue Mass Spectrometry-Based Quantitative Proteomics)

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13 pages, 335 KiB

Open AccessArticle

Validation of De Novo Peptide Sequences with Bottom-Up Tag Convolution

by Kira Vyatkina

Proteomes 2022, 10(1), 1; https://doi.org/10.3390/proteomes10010001 - 29 Dec 2021

Cited by 2 | Viewed by 2543

Abstract

De novo sequencing is indispensable for the analysis of proteins from organisms with unknown genomes, novel splice variants, and antibodies. However, despite a variety of methods developed to this end, distinguishing between the correct interpretation of a mass spectrum and a number of [...] Read more.

De novo sequencing is indispensable for the analysis of proteins from organisms with unknown genomes, novel splice variants, and antibodies. However, despite a variety of methods developed to this end, distinguishing between the correct interpretation of a mass spectrum and a number of incorrect alternatives often remains a challenge. Tag convolution is computed for a set of peptide sequence tags of a fixed length k generated from the input tandem mass spectra and can be viewed as a generalization of the well-known spectral convolution. We demonstrate its utility for validating de novo peptide sequences by using a set of those generated by the algorithm PepNovo+ from high-resolution bottom-up data sets for carbonic anhydrase 2 and the Fab region of alemtuzumab and indicate its further potential applications. Full article

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Proteomes, Volume 10, Issue 1 (March 2022) – 9 articles

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