Comprehensive discovery of the accessible primary amino group containing segments from cell surface proteins by fine-tuning high-throughput biotinylation method

ABSTRACT: Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic potential. 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 labelled extracellular peptides selectively and in larger quantities. These include the following: efficiency of the cell surface labelling, reducing the endogen protease activity by a well-controlled membrane preparation process, applying an optimal amount of affinity column for labelled peptide enrichment including adjustment of the elution steps, 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 2000 surface-associated individual labelled peptides (~6-7000 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 peptide segments that could be potential targets in various bioinformatics and molecular biology research.

INSTRUMENT(S): maXis II

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Lilla Turiak   Tamas Lango  

PROVIDER: MSV000090149 | MassIVE | Thu Aug 18 03:13:00 BST 2022

SECONDARY ACCESSION(S): PXD036143

REPOSITORIES: MassIVE