Mass spectrometry-based single cell proteomic analysis of primary human neutrophils

Introduction:

Neutrophils are vital immune cells that possess an arsenal of antimicrobial mechanisms, enabling them to kill pathogens but also damage healthy tissue if misused. They have been described as heterogeneous with direct involvement in human diseases ranging from respiratory disease to cancer. They are produced in the bone marrow, where they egress as with little transcriptional capacity, making scRNASeq poor at characterising them. Thus, a single cell proteomic (SCP) analysis would provide a much closer link to their phenotypes and functional capacities.

Methods:

Single neutrophils were sorted into 384 well plates using flow cytometry. They were frozen and later centrifuged at 300xg for 3 min before placing them in the CellenONE X1 at 50°C and 85% humidity, followed by incubation for 2h at 50°C at 85% relative humidity. Samples were kept hydrated every 15 min by addition of 500 nL water. 30 min post incubation, 500nL of 3ng/μL trypsin are added. After lysis and digestion, 2.5 μL of 0.1% TFA with 5% DMSO were added for quenching and storage.

Samples were analysed on an Orbitrap Astral, using the Vanquish Neo UHPLC with an Aurora Ultimate TS25cm nanoflow column with a 50 SPD method. The MS1 spectra were recorded at 240k res. from m/z 400-900 with AGC target of 500% and a max. injection time of 100 ms. MS2 in DIA mode used the Astral analyzer, with non-overlapping isolation windows of m/z 20 and a scan range from m/z 400–800. The precursor accumulation time set to 60ms and the AGC target to 800%. Raw files were searched on Spectronaut18.

Results:

>1,250 proteins were identified within a single neutrophil, > tripling 10x scRNASeq coverage. The increased depth enabled the mapping of the prime metabolic enzymes, and the study of cell signalling components, including vital pattern recognition receptors like TLR2, important kinases like SYK, adaptor vital adaptor proteins like FCER1G and phosphatases like PTPN6. It has also enabled a comprehensive coverage of the main neutrophil granules.

This data has enabled the study of the heterogeneity within cells, validating the differences in important markers like CD177 and showing the consistency of some of the granules proteins like ELANE. It also enabled the discovery of a neutrophil population that displayed significant reductions in the abundance of the vital granules as well as the rate limiting metabolic enzymes.

Conclusions:

By using SCP, we have identified >1,250 proteins per cell, enabling an expanded coverage of neutrophil functions at a single cell level. The data mapped the main metabolic pathways, important receptors that respond to tissue damage and infections, signalling modulators as well as important granule proteins. This work empowers the study of heterogeneity across neutrophils inter and intra individuals, enabling a better mapping neutrophil functional capacities and phenotypes and help to discover new populations and target those that are associated with disease and disfunction.