Protocol for cell surface biotinylation of magnetic labeled and captured human peripheral blood mononuclear cells

Serife Ayaz-Guner; Mustafa Burak Acar; Dudu Boyvat; Huseyin Guner; Habibe Bozalan; Melis Güzel; Selin Kübra Yıldır; Nilay Altınsoy; Fatma Fındık; Musa Karakükçü; Servet Özcan

doi:10.1016/j.xpro.2022.101863

Protocol for cell surface biotinylation of magnetic labeled and captured human peripheral blood mononuclear cells

STAR Protoc. 2022 Dec 16;3(4):101863. doi: 10.1016/j.xpro.2022.101863. Epub 2022 Nov 21.

Authors

Affiliations

¹ Department of Molecular Biology and Genetics, Izmir Institute of Technology, Izmir 35430, Türkiye; Department of Molecular Biology and Genetics, Faculty of Life and Natural Science, Abdullah Gül University, Kayseri 38080, Türkiye.
² Faculty of Science, Department of Biology, Erciyes University, Kayseri 38039, Türkiye; Genome and Stem Cell Center (GENKÖK), Erciyes University, Kayseri 38039, Türkiye.
³ Bioengineering Program, Graduate School of Engineering and Science, Abdullah Gul University, Kayseri 38080, Türkiye.
⁴ Department of Molecular Biology and Genetics, Faculty of Life and Natural Science, Abdullah Gül University, Kayseri 38080, Türkiye.
⁵ Genome and Stem Cell Center (GENKÖK), Erciyes University, Kayseri 38039, Türkiye; Gevher Nesibe Genome and Stem Cell Institute, Erciyes University, Kayseri 38039, Türkiye.
⁶ Genome and Stem Cell Center (GENKÖK), Erciyes University, Kayseri 38039, Türkiye; Institute of Health Sciences, Erciyes University, Kayseri 38039, Türkiye.
⁷ Department of Molecular Biology and Genetics, Izmir Institute of Technology, Izmir 35430, Türkiye.
⁸ KANKA Pediatric Bone Marrow Transplantation Center, Department of Pediatric Hematology and Oncology, Faculty of Medicine, Erciyes University, Kayseri 38039, Türkiye.
⁹ Faculty of Science, Department of Biology, Erciyes University, Kayseri 38039, Türkiye; Genome and Stem Cell Center (GENKÖK), Erciyes University, Kayseri 38039, Türkiye. Electronic address: ozcan@erciyes.edu.tr.

Abstract

Analysis of the surfaceome of a blood cell subset requires cell sorting, followed by surface protein enrichment. Here, we present a protocol combining magnetically activated cell sorting (MACS) and surface biotinylation of the target cell subset from human peripheral blood mononuclear cells (PBMCs). We describe the steps for isolating target cells and their in-column surface biotinylation, followed by isolation and mass spectrometry analysis of biotinylated proteins. The protocol enables in-column surface biotinylation of specific cell subsets with minimal membrane disruption.

Keywords: Cell Biology; Cell Isolation; Mass Spectrometry; Protein Biochemistry; Proteomics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biotinylation
Cell Membrane / metabolism
Humans
Leukocytes, Mononuclear* / chemistry
Magnetic Phenomena
Membrane Proteins* / metabolism

Substances

Membrane Proteins