Cargando…

Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method

Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of...

Descripción completa

Detalles Bibliográficos
Autores principales: Langó, Tamás, Kuffa, Katalin, Tóth, Gábor, Turiák, Lilla, Drahos, László, Tusnády, Gábor E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9820203/
https://www.ncbi.nlm.nih.gov/pubmed/36613715
http://dx.doi.org/10.3390/ijms24010273
Descripción
Sumario:Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of the cell surface subproteome of individual cells. Here we present a high-throughput technique which relies on the biotinylation of the accessible primary amino groups in the extracellular segments of the proteins, using HL60 as a model cell line. Several steps of the method have been thoroughly optimized to capture labeled surface proteins selectively and in larger quantities. These include the following: improving the efficiency of the cell surface biotinylation; reducing the endogen protease activity; applying an optimal amount of affinity column and elution steps for labeled peptide enrichment; and examining the effect of various solid-phase extraction methods, different HPLC gradients, and various tandem mass spectrometry settings. Using the optimized workflow, we identified at least 1700 surface-associated individual labeled peptides (~6000–7000 redundant peptides) from the model cell surface in a single nanoHPLC-MS/MS run. The presented method can provide a comprehensive and specific list of the cell surface available protein segments that could be potential targets in various bioinformatics and molecular biology research.