The Single Cell Proteomic blueprint, navigating instrumentation platforms, software tools and high-load libraries in neutrophils, RKO and A549 cells

Brenes, A. J.; Mayer, R. L.; Makar, A.; Coelho, P.; van Stralen, G.; Sadiku, P.; Walmsley, S. R.; Matzinger, M.; Mechtler, K.; von Kriegsheim, A.

doi:10.64898/2026.06.12.731618

bioRxiv · 10.64898/2026.06.12.731618

The Single Cell Proteomic blueprint, navigating instrumentation platforms, software tools and high-load libraries in neutrophils, RKO and A549 cells

Brenes, A. J.·Mayer, R. L.·Makar, A.·Coelho, P.·van Stralen, G.·Sadiku, P.·Walmsley, S. R.·Matzinger, M.·Mechtler, K.·von Kriegsheim, A.

View original source ↗Export metadata ↓

Abstract

Mass spectrometry-based single cell proteomics (SCP) is rapidly emerging as a powerful approach for biological research, with applications extending beyond in-vitro cancer cell lines. Recent advances make it possible to apply SCP to ex-vivo human cells from tissues such as the brain and pancreas, as well as to technically challenging immune populations such as neutrophils. However, these analyses remain more challenging and typically result in reduced proteomic coverage. To support the development of robust workflows for SCP data acquisition and analysis, we systematically evaluated multiple DIA search engines, search engine settings, the inclusion of high-load library samples in single-cell search spaces, the impact of contaminants, and the quantitative properties of identified proteins. These comparisons were performed across two major instrumentation platforms, Orbitrap Astral and timsTOF SCP, and across A549, RKO cells and neutrophils, three cell types differing in size and protein content. Our work here provides guidelines on the software parameters to use for SCP, instrument specific results and cell dependent optimizations of high-load libraries, as well as novel evaluation of the quantitative properties of proteins for single cell and low input proteomics.

Explore related subjects

biochemistry Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Brenes, A. J., Mayer, R. L., Makar, A., Coelho, P., van Stralen, G., Sadiku, P., Walmsley, S. R., Matzinger, M., Mechtler, K., von Kriegsheim, A.. 2026-06-16. The Single Cell Proteomic blueprint, navigating instrumentation platforms, software tools and high-load libraries in neutrophils, RKO and A549 cells. https://doi.org/10.64898/2026.06.12.731618

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Impacts of Environmental Nickel and Chromium Exposure on the Blood Proteome of House Sparrows (Passer domesticus) in New Caledonia

New Caledonia holds some of the world's largest ultramafic mineral deposits, and nickel (Ni) smelting operations have created a gradient of Ni and chromium (Cr) contamination across the archipelago. Molecular impacts of Ni and Cr toxicity on wildlife remain largely unexplored. Here, we used the house sparrow (Passer domesticus) as a sentinel species to characterise the blood proteomic response to Ni and Cr exposure near the Doniambo smelter in Noumea. Blood was collected from 52 free-living sparrows across 18 sites classified by soil metal concentration. Plasma and blood cell pellet fractions were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma proteomics identified histidine-rich glycoprotein (HRG) as the most prominent responder, with approximately 2.1-fold elevation in exposed relative to low-exposure birds. HRG contains a conserved histidine-rich region that binds cations including divalent nickel, and paired plasma-liver analysis confirmed hepatic-driven synthesis. Blood cell pellet proteomics revealed that Ni-Cr co-exposure predominantly disrupts the nuclear compartment: histone depletion indicated chromatin collapse, NUP153 dysregulation impaired nuclear pore function, and VRK1/TRIP13 upregulation suggested DNA damage response activation coupled with mitotic checkpoint override. Concurrent RNA processing collapse (DDX17, DHX15, RBMX, ALYREF) and changes in telomere-associated proteins (TERF2IP, hnRNPA1 and VRK1) implicated disrupted genome maintenance. These findings identify HRG as a candidate plasma biomarker of Ni-Cr exposure and reveal a coordinated nuclear toxicity signature linking heavy metal contamination to DNA damage and epigenetic disruption in a free-living vertebrate.

biochemistry↗

bioRxiv2026

Programmable amide bond formation using de novo ligases

Amide bond formation is of central importance in the chemical industry, underpinning the synthesis of small bioactive molecules, macrocycles, peptides and their conjugates. Conventional methods of amide synthesis rely on stoichiometric coupling agents and costly protecting group manipulations. While biocatalysis can alleviate some of these challenges, enzymes typically operate across a narrow range of substrates and their adaptation to new coupling partners requires extensive engineering. Here we show that de novo amide ligases featuring a cysteine catalytic triad can be programmed computationally to operate across diverse substrate classes with high selectivity. We first designed complementary peptide ligases to selectively functionalise either the N-terminus or a single lysine residue of a polypeptide sequence containing 36 competing lysines. Crystal structures of both ligases show excellent agreement with the designs, with structural modelling suggesting that amidation outcompetes hydrolysis because the catalytically competent geometry forms only upon acceptor binding. We then extended our approach to the synthesis of small bioactive molecules by designing stereoselective ligases that mediate a key amide bonding forming step in the synthesis of the preventative HIV therapeutic lenacapavir. These results establish de novo enzyme design as a programmable platform for achieving selective amide bond formations across a wide range of molecular targets.

biochemistry↗

bioRxiv2026

EngA adopts multiple distinct conformational states during ribosome assembly

Ribosome assembly, a highly coordinated and crucial cellular process, is assisted by various assembly factors, including GTPases. EngA, an essential P-loop GTPase, is involved in late-stage assembly of the 50S ribosomal subunit. The structures of 50S ribosome in complex with EngA-bound GMPPNP, and EngA-bound GDP alone, without 50S subunit, have been reported previously. However, the molecular mechanism of EngA GTP hydrolysis-assisted 50S maturation remains poorly understood, and structures of the 50S ribosome in complex with EngA-bound GDP have yet to be deciphered. We have reported cryo- EM structures of the Mycobacterium smegmatis 50S ribosome in complex with EngA-bound GTP{gamma}S or GDP at 2.8 - 3.5 [A] resolution. Structural analysis revealed that EngA and 50S subunit undergo a coordinated structural rearrangement, and an assembly intermediate is formed in which the stem-loop of H80 makes base-pair interactions with the junction of H89 and H90, forming a unique stem-loop-junction (SLJ) motif. EngA-bound GDP adopts distinctly different conformations in its ribosome-bound and unbound states. In summary, this study proposed a multi-state conformational model for EngA-assisted ribosome assembly.

biochemistry↗