2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications

Kendrick, Nancy C.; Darie, Costel C.; Hoelter, Matt; Powers, Ginny; Johansen, Jon

doi:10.1007/978-3-030-15950-4_33

Cited by 12 publications

(11 citation statements)

References 19 publications

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“…Its separating resolution is higher than size exclusion chromatography, but not as high as 2D-PAGE. 2D-PAGE has a high resolution and, with the introduction of immobilized pH gradient (IPG) strips, its reproducibility has improved considerably, becoming the method of choice for most proteoform studies [ 46 , 47 ]. The development of PTM-specific stains for phosphoproteins (e.g., Pro-Q Diamond) and glycoproteins (e.g., Pro-Q Emerald, Dansylhydrazine) further increased the applicability of 2D-PAGE [ 48 ].…”

Section: Sample Preparationmentioning

confidence: 99%

A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field

et al. 2020

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Proteomics is the field of study that includes the analysis of proteins, from either a basic science prospective or a clinical one. Proteins can be investigated for their abundance, variety of proteoforms due to post-translational modifications (PTMs), and their stable or transient protein–protein interactions. This can be especially beneficial in the clinical setting when studying proteins involved in different diseases and conditions. Here, we aim to describe a bottom-up proteomics workflow from sample preparation to data analysis, including all of its benefits and pitfalls. We also describe potential improvements in this type of proteomics workflow for the future.

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Section: Sample Preparationmentioning

confidence: 99%

A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field

et al. 2020

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“…In the post-genome era, research into proteomics provides the opportunity to analyze thousands of proteins in complex mixtures. Combining the 2D-PAGE and mass spectrometry techniques has become a powerful methodology that allows the identification of protein spots of interest [ 38 ]. We implemented this proteomic approach to obtain the OMP profiles of the seven strains defined as AIEC and then compare them with the OMP profile of the commensal E. coli HS strain.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Adherent-Invasive Escherichia coli (AIEC) Outer Membrane Proteins Provides Potential Molecular Markers to Screen Putative AIEC Strains

Saitz

Rasko

Pardo

et al. 2022

IJMS

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Adherent-invasive E. coli (AIEC) is a pathotype associated with the etiopathogenesis of Crohn’s disease (CD), albeit with an as-yet unclear role. The main pathogenic mechanisms described for AIEC are adherence to epithelial cells, invasion of epithelial cells, and survival and replication within macrophages. A few virulence factors have been described as participating directly in these phenotypes, most of which have been evaluated only in AIEC reference strains. To date, no molecular markers have been identified that can differentiate AIEC from other E. coli pathotypes, so these strains are currently identified based on the phenotypic characterization of their pathogenic mechanisms. The identification of putative AIEC molecular markers could be beneficial not only from the diagnostic point of view but could also help in better understanding the determinants of AIEC pathogenicity. The objective of this study was to identify molecular markers that contribute to the screening of AIEC strains. For this, we characterized outer membrane protein (OMP) profiles in a group of AIEC strains and compared them with the commensal E. coli HS strain. Notably, we found a set of OMPs that were present in the AIEC strains but absent in the HS strain. Moreover, we developed a PCR assay and performed phylogenomic analyses to determine the frequency and distribution of the genes coding for these OMPs in a larger collection of AIEC and other E. coli strains. As result, it was found that three genes (chuA, eefC, and fitA) are widely distributed and significantly correlated with AIEC strains, whereas they are infrequent in commensal and diarrheagenic E. coli strains (DEC). Additional studies are needed to validate these markers in diverse strain collections from different geographical regions, as well as investigate their possible role in AIEC pathogenicity.

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“…However, it also has several disadvantages that make it of little value in the search of possible cancer biomarkers: it is challenging to automate; very large or hydrophobic proteins do not enter the gel during the first-dimension electrophoresis, while very acidic or fundamental proteins are not well resolved (for example, the proteins of cell membranes, which represent 40% of all cell proteins). It also struggles to detect rare proteins, some of which are very important (regulatory proteins, proteins involved in signal transduction, receptors) [57][58][59].…”

Section: Two-dimensional Gels Electrophoresismentioning

confidence: 99%

Understanding Cervical Cancer through Proteomics

Martínez-Rodríguez

Limones-González

Mendoza-Almanza

et al. 2021

Cells

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Cancer is one of the leading public health issues worldwide, and the number of cancer patients increases every day. Particularly, cervical cancer (CC) is still the second leading cause of cancer death in women from developing countries. Thus, it is essential to deepen our knowledge about the molecular pathogenesis of CC and propose new therapeutic targets and new methods to diagnose this disease in its early stages. Differential expression analysis using high-throughput techniques applied to biological samples allows determining the physiological state of normal cells and the changes produced by cancer development. The cluster of differential molecular profiles in the genome, the transcriptome, or the proteome is analyzed in the disease, and it is called the molecular signature of cancer. Proteomic analysis of biological samples of patients with different grades of cervical intraepithelial neoplasia (CIN) and CC has served to elucidate the pathways involved in the development and progression of cancer and identify cervical proteins associated with CC. However, several cervical carcinogenesis mechanisms are still unclear. Detecting pathologies in their earliest stages can significantly improve a patient’s survival rate, prognosis, and recurrence. The present review is an update on the proteomic study of CC.

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2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications

Cited by 12 publications

References 19 publications

A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field

A Critical Review of Bottom-Up Proteomics: The Good, the Bad, and the Future of This Field

Characterization of Adherent-Invasive Escherichia coli (AIEC) Outer Membrane Proteins Provides Potential Molecular Markers to Screen Putative AIEC Strains

Understanding Cervical Cancer through Proteomics

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