Titel (eng)

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection

Autor*in

Huriye Ercan   Medical University of Vienna / Immunology Outpatient Clinic

Maria Zellner   Medical University of Vienna

Ingrid Miller   University of Veterinary Medicine, Vienna

Margarethe Geiger   Medical University of Vienna

Jae-Won Yang   Medical University of Vienna

Christopher Gerner   University of Vienna

Andrea Bileck   University of Vienna

Goran Mitulovic   Medical University of Vienna

Ulrike Resch   Medical University of Vienna

Felicia Hsu   Medical University of Vienna

Verlag

MDPI

Beschreibung (eng)

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

Sprache des Objekts

Englisch

Datum

2023

Rechte

Dieses Werk bzw. dieser Inhalt steht unter einer
CC BY 4.0 - Creative Commons Namensnennung 4.0 International Lizenz.

CC BY 4.0 International

http://creativecommons.org/licenses/by/4.0/

Klassifikation

Label-Free Quantification; 2-Dimensional Electrophoresis; Biological Variation; Alzheimers-Disease; Apolipoprotein-E; 2-D Dige; Protein; Shotgun; Identification; Purification

Mitglied in der/den Collection(s) (1)