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MS-Based Protein Specific Analysis

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Macromolecules".

Deadline for manuscript submissions: closed (28 February 2022) | Viewed by 25912

Special Issue Editor


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Guest Editor
Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
Interests: mass spectrometry; hyphenation; separation techniques; native protein analysis; affinity separations; automatization; protein characterization; biopharmaceuticals; clinical analysis

Special Issue Information

Dear Colleagues,

Proteins are essential macromolecules required in the structure, function, and regulation of many processes in living organisms. Proteins are very diverse and often comprise genetic variants and posttranslational modifications (i.e., proteoforms). Assessing protein and proteoform variability is essential – yet challenging – in many research areas, including medical, biological, and chemical sciences. Mass spectrometry (MS) has become the primary tool for the structural characterization of proteins and many different MS-based approaches – from bottom-up to intact and top-down approaches – are currently being used for protein identification and proteoform characterization. Often, upfront separation of proteins is necessary to unravel the vast heterogeneity and obtain accurate mass information. Thanks to recent technological developments in MS instrumentation, separation technology, and coupling options, online approaches are gaining a predominant position in the MS toolbox for protein analysis.

This Special Issue will focus on MS-based protein analysis and will cover recent methodological developments and relevant applications. Applications may cover characterization of novel endogenous or recombinant proteins, biomarker discovery, or biologically relevant proteomic studies, among others. Original research work, communications as well as reviews in the field are welcome to this Special Issue.

Dr. Elena Domínguez-Vega
Guest Editor

Manuscript Submission Information

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Keywords

  • Mass spectrometry
  • Top-down proteomics
  • Bottom-up proteomics
  • Intact protein analysis
  • Middle-up/down characterization
  • Hyphenation
  • Post-translational modifications
  • Proteoforms
  • Endogenous proteins
  • Biopharmaceuticals
  • Industrial enzymes
  • Clinical proteomics

Published Papers (8 papers)

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Research

20 pages, 3318 KiB  
Article
Plasma Proteomic Profile of Patients with Tick-Borne Encephalitis and Co-Infections
by Agnieszka Gęgotek, Anna Moniuszko-Malinowska, Monika Groth, Sławomir Pancewicz, Piotr Czupryna, Justyna Dunaj, Sinemyiz Atalay, Piotr Radziwon and Elżbieta Skrzydlewska
Int. J. Mol. Sci. 2022, 23(8), 4374; https://doi.org/10.3390/ijms23084374 - 15 Apr 2022
Cited by 4 | Viewed by 2011
Abstract
Despite the increasing number of patients suffering from tick-borne encephalitis (TBE), Lyme disease, and their co-infection, the mechanisms of the development of these diseases and their effects on the human body are still unknown. Therefore, the aim of this study was to evaluate [...] Read more.
Despite the increasing number of patients suffering from tick-borne encephalitis (TBE), Lyme disease, and their co-infection, the mechanisms of the development of these diseases and their effects on the human body are still unknown. Therefore, the aim of this study was to evaluate the changes in the proteomic profile of human plasma induced by the development of TBE and to compare it with changes in TBE patients co-infected with other tick-borne pathogens. The results obtained by proteomic analysis using a nanoLC-Q Exactive HF mass spectrometer showed that the most highly elevated groups of proteins in the plasma of TBE patients with co-infection were involved in the pro-inflammatory response and protein degradation, while the antioxidant proteins and factors responsible for protein biosynthesis were mainly downregulated. These results were accompanied by enhanced GSH- and 4-HNE-protein adducts formation, observed in TBE and co-infected patients at a higher level than in the case of patients with only TBE. In conclusion, the differences in the proteomic profiles between patients with TBE and co-infected patients indicate that these diseases are significantly diverse and, consequently, require different treatment, which is particularly important for further research, including the development of novel diagnostics tools. Full article
(This article belongs to the Special Issue MS-Based Protein Specific Analysis)
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23 pages, 4160 KiB  
Article
Proteomic Analysis Reveals Enzymes for β-D-Glucan Formation and Degradation in Levilactobacillus brevis TMW 1.2112
by Julia A. Bockwoldt, Chen Meng, Christina Ludwig, Michael Kupetz and Matthias A. Ehrmann
Int. J. Mol. Sci. 2022, 23(6), 3393; https://doi.org/10.3390/ijms23063393 - 21 Mar 2022
Cited by 3 | Viewed by 2266
Abstract
Bacterial exopolysaccharide (EPS) formation is crucial for biofilm formation, for protection against environmental factors, or as storage compounds. EPSs produced by lactic acid bacteria (LAB) are appropriate for applications in food fermentation or the pharmaceutical industry, yet the dynamics of formation and degradation [...] Read more.
Bacterial exopolysaccharide (EPS) formation is crucial for biofilm formation, for protection against environmental factors, or as storage compounds. EPSs produced by lactic acid bacteria (LAB) are appropriate for applications in food fermentation or the pharmaceutical industry, yet the dynamics of formation and degradation thereof are poorly described. This study focuses on carbohydrate active enzymes, including glycosyl transferases (GT) and glycoside hydrolases (GH), and their roles in the formation and potential degradation of O2-substituted (1,3)-β-D-glucan of Levilactobacillus (L.) brevis TMW 1.2112. The fermentation broth of L. brevis TMW 1.2112 was analyzed for changes in viscosity, β-glucan, and D-glucose concentrations during the exponential, stationary, and early death phases. While the viscosity reached its maximum during the stationary phase and subsequently decreased, the β-glucan concentration only increased to a plateau. Results were correlated with secretome and proteome data to identify involved enzymes and pathways. The suggested pathway for β-glucan biosynthesis involved a β-1,3 glucan synthase (GT2) and enzymes from maltose phosphorylase (MP) operons. The decreased viscosity appeared to be associated with cell lysis as the β-glucan concentration did not decrease, most likely due to missing extracellular carbohydrate active enzymes. In addition, an operon was discovered containing known moonlighting genes, all of which were detected in both proteome and secretome samples. Full article
(This article belongs to the Special Issue MS-Based Protein Specific Analysis)
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16 pages, 2250 KiB  
Article
ABPP-HT*—Deep Meets Fast for Activity-Based Profiling of Deubiquitylating Enzymes Using Advanced DIA Mass Spectrometry Methods
by Hannah B. L. Jones, Raphael Heilig, Simon Davis, Roman Fischer, Benedikt M. Kessler and Adán Pinto-Fernández
Int. J. Mol. Sci. 2022, 23(6), 3263; https://doi.org/10.3390/ijms23063263 - 17 Mar 2022
Cited by 4 | Viewed by 3364
Abstract
Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging [...] Read more.
Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow, we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling) that in combination with DIA MS methods, allowed for the consistent profiling of 35–40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day. Full article
(This article belongs to the Special Issue MS-Based Protein Specific Analysis)
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15 pages, 1761 KiB  
Article
Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms
by Guusje van Schaick, Nadi el Hajjouti, Simone Nicolardi, Joost den Hartog, Romana Jansen, Rob van der Hoeven, Wim Bijleveld, Nicolas Abello, Manfred Wuhrer, Maurien M. A. Olsthoorn and Elena Domínguez-Vega
Int. J. Mol. Sci. 2022, 23(3), 1307; https://doi.org/10.3390/ijms23031307 - 24 Jan 2022
Cited by 4 | Viewed by 2772
Abstract
Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the [...] Read more.
Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms. Full article
(This article belongs to the Special Issue MS-Based Protein Specific Analysis)
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14 pages, 3276 KiB  
Article
Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells
by Lisa Strasser, Stefano Boi, Felipe Guapo, Nicholas Donohue, Niall Barron, Alana Rainbow-Fletcher and Jonathan Bones
Int. J. Mol. Sci. 2021, 22(21), 11499; https://doi.org/10.3390/ijms222111499 - 25 Oct 2021
Cited by 13 | Viewed by 5574
Abstract
Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host [...] Read more.
Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing. Full article
(This article belongs to the Special Issue MS-Based Protein Specific Analysis)
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17 pages, 3549 KiB  
Article
Mass Spectrometry-Based Proteomics Reveal Alcohol Dehydrogenase 1B as a Blood Biomarker Candidate to Monitor Acetaminophen-Induced Liver Injury
by Floriane Pailleux, Pauline Maes, Michel Jaquinod, Justine Barthelon, Marion Darnaud, Claire Lacoste, Yves Vandenbrouck, Benoît Gilquin, Mathilde Louwagie, Anne-Marie Hesse, Alexandra Kraut, Jérôme Garin, Vincent Leroy, Jean-Pierre Zarski, Christophe Bruley, Yohann Couté, Didier Samuel, Philippe Ichai, Jamila Faivre and Virginie Brun
Int. J. Mol. Sci. 2021, 22(20), 11071; https://doi.org/10.3390/ijms222011071 - 14 Oct 2021
Cited by 2 | Viewed by 2460
Abstract
Acute liver injury (ALI) is a severe disorder resulting from excessive hepatocyte cell death, and frequently caused by acetaminophen intoxication. Clinical management of ALI progression is hampered by the dearth of blood biomarkers available. In this study, a bioinformatics workflow was developed to [...] Read more.
Acute liver injury (ALI) is a severe disorder resulting from excessive hepatocyte cell death, and frequently caused by acetaminophen intoxication. Clinical management of ALI progression is hampered by the dearth of blood biomarkers available. In this study, a bioinformatics workflow was developed to screen omics databases and identify potential biomarkers for hepatocyte cell death. Then, discovery proteomics was harnessed to select from among these candidates those that were specifically detected in the blood of acetaminophen-induced ALI patients. Among these candidates, the isoenzyme alcohol dehydrogenase 1B (ADH1B) was massively leaked into the blood. To evaluate ADH1B, we developed a targeted proteomics assay and quantified ADH1B in serum samples collected at different times from 17 patients admitted for acetaminophen-induced ALI. Serum ADH1B concentrations increased markedly during the acute phase of the disease, and dropped to undetectable levels during recovery. In contrast to alanine aminotransferase activity, the rapid drop in circulating ADH1B concentrations was followed by an improvement in the international normalized ratio (INR) within 10–48 h, and was associated with favorable outcomes. In conclusion, the combination of omics data exploration and proteomics revealed ADH1B as a new blood biomarker candidate that could be useful for the monitoring of acetaminophen-induced ALI. Full article
(This article belongs to the Special Issue MS-Based Protein Specific Analysis)
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17 pages, 3460 KiB  
Article
Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics
by Fiammetta Di Marco, Thomas Berger, Wolfgang Esser-Skala, Erdmann Rapp, Christof Regl and Christian G. Huber
Int. J. Mol. Sci. 2021, 22(16), 9072; https://doi.org/10.3390/ijms22169072 - 23 Aug 2021
Cited by 12 | Viewed by 3552
Abstract
Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of [...] Read more.
Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes. Full article
(This article belongs to the Special Issue MS-Based Protein Specific Analysis)
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22 pages, 6117 KiB  
Article
Proteomic Profiling of Ectosomes Derived from Paired Urothelial Bladder Cancer and Normal Cells Reveals the Presence of Biologically-Relevant Molecules
by Magdalena Surman, Sylwia Kędracka-Krok, Urszula Jankowska, Anna Drożdż, Ewa Stępień and Małgorzata Przybyło
Int. J. Mol. Sci. 2021, 22(13), 6816; https://doi.org/10.3390/ijms22136816 - 24 Jun 2021
Cited by 4 | Viewed by 2683
Abstract
Protein content of extracellular vesicles (EVs) can modulate different processes during carcinogenesis. Novel proteomic strategies have been applied several times to profile proteins present in exosomes released by urothelial bladder cancer (UBC) cells. However, similar studies have not been conducted so far on [...] Read more.
Protein content of extracellular vesicles (EVs) can modulate different processes during carcinogenesis. Novel proteomic strategies have been applied several times to profile proteins present in exosomes released by urothelial bladder cancer (UBC) cells. However, similar studies have not been conducted so far on another population of EVs, i.e., ectosomes. In the present study we used a shotgun nanoLC–MS/MS proteomic approach to investigate the protein content of ectosomes released in vitro by T-24 UBC cells and HCV-29 normal ureter epithelial cells. In addition, cancer-promoting effects exerted by UBC-derived ectosomes on non-invasive cells in terms of cell proliferation and migratory properties were assessed. In total, 1158 proteins were identified in T-24-derived ectosomes, while HCV-29-derived ectosomes contained a lower number of 259 identified proteins. Qualitative analysis revealed 938 proteins present uniquely in T-24-derived ectosomes, suggesting their potential applications in bladder cancer management as diagnostic and prognostic biomarkers. In addition, T-24-derived ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of the identified cancer-promoting molecules. The present study provided a focused identification of biologically relevant proteins in UBC-derived ectosomes, confirming their role in UBC development and progression, and their applicability for further biomarker-oriented studies in preclinical or clinical settings. Full article
(This article belongs to the Special Issue MS-Based Protein Specific Analysis)
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