bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.01.05.574392

Contaminant Spot Check and Removal Assay (ContamSPOT) for Mass Spectrometry Analysis

Smeriglio, N.·Li, H.·binti Mazli, W. N. A.·Bendel, K. B.·Hao, L.
View original source ↗Export metadata ↓

Abstract

Mass spectrometry (MS) analysis is often challenged by contaminations from detergents, salts, and polymers that compromise the data quality and can damage the chromatography and mass spectrometry instruments. However, researchers may not discover contamination issues until after data acquisition. There is no existing contaminant assay that is sensitive enough to detect trace amounts of contaminants from a few microliters of samples prior to MS analysis. To address this crucial need in the field, we developed a sensitive, rapid, and low-cost contaminant spot check and removal assay (ContamSPOT) to detect and quantify trace amounts of contaminants, such as detergents, salts, and other chemicals commonly used in MS sample preparation workflow. Only one microliter of sample was used prior to MS injection to quantify contaminants by ContamSPOT colorimetric or fluorometric assay on a thin layer chromatography (TLC) plate. We also optimized contaminant removal methods to salvage samples with minimal loss when ContamSPOT showed a positive result. ContamSPOT was then successfully applied to evaluate commonly used bottom- up proteomic methods, regarding the effectiveness of removing detergents, peptide recovery, and reproducibility. We expect ContamSPOT to be widely adopted by MS laboratories as a last-step quality checkpoint prior to MS injection. ContamSPOT can also provide a unique readout of sample cleanness for developing and evaluating MS-based sample preparation methods. We provided a practical decision tree and a step-by-step protocol with a troubleshooting guide to facilitate the use of ContamSPOT by other researchers. Table of Contents (TOC) graphic(All images used in the TOC graphic are original, in compliance with copyright guidelines.) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/574392v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@7b6f0aorg.highwire.dtl.DTLVardef@1b6fcd9org.highwire.dtl.DTLVardef@9213a5org.highwire.dtl.DTLVardef@47b7fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Smeriglio, N., Li, H., binti Mazli, W. N. A., Bendel, K. B., Hao, L.. 2024-01-06. Contaminant Spot Check and Removal Assay (ContamSPOT) for Mass Spectrometry Analysis. https://doi.org/10.1101/2024.01.05.574392

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

KEEP EXPLORING

Related preprints

bioRxiv2026

aaRSID, an engineered pyrrolysyl-tRNA synthetase platform for multi-probe proximity proteomics

Proximity labeling (PL) methods utilize spatially targeted chemical or enzymatic generation of a diffusible, reactive intermediate to covalently tag neighboring proteins in living systems. Unlike other tools for studying molecular interactions, PL can detect transient protein relationships with high spatial and temporal sensitivity, allowing for insight into their roles in biological processes. However, current enzymatic PL tools, such as TurboID and APEX2, are limited by their substrate structure and chemistry, which can generate significant background and/or perturb cellular physiology. To address these limitations, we have developed aminoacyl-tRNA synthetase ID (aaRSID), a PL tool that leverages an engineered pyrrolysyl tRNA synthetase (PylRS) for proximity labeling of proteins. We chose PylRS because it can catalyze promiscuous lysine labeling in the absence of its cognate tRNA and utilize a variety of non-canonical amino acids (ncAAs) as substrates. Here, we demonstrate aaRSID's intrinsic proximity labeling activity, use directed evolution to improve this activity, and apply the improved mutant (aaRSID-Ma1.3) for subcellular proteomics and multiplexed imaging. Our work establishes aminoacyl-tRNA synthetases as a new PL enzyme class and introduces a versatile chemical platform for developing ncAA-derived probes to map cellular microenvironments, greatly expanding the applications possible of PL technology.

biochemistry↗
bioRxiv2026

Cellular uptake of folate-olaparib conjugates via folate receptor-mediated endocytosis: Potential for selective delivery of DNA damage response inhibitors into tumour cells

The folate receptor (FR) is overexpressed in a range of human tumours including ovarian cancer cells. We propose that the overexpression of the FR on the surface of ovarian tumour cells could be exploited for the selective delivery of a DNA damage response inhibitor (DDRi) in the form of an intact folate drug conjugate (FDC). This approach would improve the therapeutic index of the parent DDRi facilitating combination studies of the DDRi-based FDC with DNA damaging chemotherapy. FR-mediated cellular uptake of the proposed folate drug conjugates is requisite for FDC selective delivery into tumours. In this study, we synthesised a series of olaparib-based folate conjugates that maintained the biochemical PARP1 inhibition associated with olaparib and showed binding affinity for the folate receptor. Significantly, we identified compounds 10b and 11 that selectively enter FR overexpressing tumour cells via folate receptor-mediated endocytosis in their intact form and engage with their target as demonstrated by the potent inhibition of PARylation (KB cells, PARylation IC50 = 5.7 and 3.9 nM; respectively).

biochemistry↗
bioRxiv2026

Architecture and Energy Transfer of the Bacterial Photosynthetic Unit

In phototrophic organisms, pigment-protein membrane complexes are densely packed to form photosynthetic units (PSUs) that capture solar energy and convert it into chemical energy. Although the structures of many individual photosynthetic complexes have been resolved, how they are arranged and interact with others within photosynthetic membranes to enable efficient excitation energy transfer (EET) remains poorly understood. Here, we report cryo-electron microscopy structures of PSU supercomplex assemblies from the phototrophic a-proteobacterium Rhodovulum viride, including an RC-LH1 core associated with one or two peripheral LH2 complexes and a curved LH2 tetramer. These membrane-derived assemblies define the relative positions and orientations of neighboring photosynthetic complexes and place their pigment arrays in proximity across antenna-antenna and antenna-core interfaces. Structure-based simulations identify potential EET pathways within the PSU assemblies and reveal rapid energy transfer across both LH2-LH2 and LH2-LH1 interfaces. Collectively, these findings provide insights into the assembly and structural modularity of bacterial PSUs and elucidate how the lateral organization of membrane protein complexes facilitates efficient energy transfer. This work extends structural studies of bacterial photosynthesis from individual complexes to their native higher-order assembly, providing a framework for understanding how photosynthetic supercomplex organization shapes energy migration and for guiding the design of artificial photosynthesis.

biochemistry↗