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Alexander Ivanov talking about increases in sensitivity and signal to noise when changing to ultra low flor rates for single cell proteomics. #ESCP2025

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The last session of the day is opened by Tami Geiger looking at the interactions of the immune cells with the cancer microenvironments using single cell proteomics #ESCP2025

Its exciting to see that its a full house at #ESCP2025 lots of enthusiastic single cell proteomics researchers 🤓

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Wilfred Rossol is next talking about proteomic profiling of protein aggregates with a more spatial proteomics approach #ESCP2025

Next up is @pedroaragon.bsky.social talking about single cell and low input proteomics to study perturbation in leukemia #ESCP2025

Up next is @jwoessmann.bsky.social from the @erwinschoof.bsky.social lab showing how to focus on the exciting lower abundance proteins with single cell proteomics #ESCP2025

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Daniela Valdes from @coscialab.bsky.social lab presents how smoking disrupts the placental function via macrophage dysregulation using single nuclei analysis and deep visual proteomics #ESCP2025

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Cool work from @nanopots.bsky.social doing cell surface profiling of 100-500 immine cells using affinity purification #ESCP2025

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Next up is @vadim-demichev.bsky.social telling us how quantitatively accurate is single cell proteomics #ESCP2025

Alejandro Brenes @ajbrenes.com · 25/08/2025

After the DVP symposium now begins the start of the European Symposium on Single Cell Proteomics #ECSP25 A quick intro from @kmechtler.bsky.social and we kick off two days of exciting science starting with SCP of neutrophils in glioblastoma

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Start of the European symposium in single cell proteomics at beautiful Viena with a workshop on spatial proteomics organised by @coscialab.bsky.social

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In summary, we do single cell proteomics of glioblastoma neutrophils and find exciting functional states invisible to scRNAseq which further define the roles that neutrophils play within glioblastomas, showing some pro-tumorigenic and anti-tumorigenic populations and properties

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Finally we find a population with a lysosomal & protein degradation signature. They have high immunoglobulins, suggesting phagocytosis. However this is not positive, as they have immunosuppresive (ARG1) & angiogenic signature. Perhaps phagocytosis reprograms to aid the tumour

Our lytic NET (LN) populations proteomic profile closely matches the @adrover.bsky.social vascular restricted population, with NETs actively causing occlusion of the vasculature leading to necrosis. Our LN data suggests the same may occur in human GBM as we they show clotting and NETs signature

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a lytic NETs population also looked vascular restricted. The proteome showed: ⬆️ coagulation & complement proteins ~ 90% ⬇️ in histones, azurophilic granules, secretory vesicles, nuclear membrane Also had ⬇️ in metabolic & cell surface proteins, suggesting they can't respond to much anymore

two populations looked vascular restricted. The first shared the immature signature identified from the mini-bulk: ⬆️ mitochondrial & ribosomal proteins ⬆️ PCNA & CEBPE ⬇️ NADPH oxidase & CD16 The important of ECM proteins, this population had a blood signature with coagulation & apolipoproteins

the vital NETs population also has ⬇️ granules, but combined with ⬇️ in almost all histones and nuclear membrane proteins. This decrease was not mirrored in the exhausted population.

data shows a trajectory where the exhausted neutrophils have degranulated without any hint of NETosis (histones are stable). But they do show marked ⬇️ in key metabolic enzymes in glycolysis, glycogenolysis and PPP. They also have ⬇️ reg. of neutrophil functions like RAC2 and calmodulin

The engaged then seem to follow a trajectory that leads to exhaustion or vital NETosis. just as a reminder neutrophil extracellular traps are mostly composed of DNA, histones and granule proteins. All invisible to RNAseq in mature neutrophils, but very much visible to proteomics

The armed population had the highest abundance of migration related proteins, combined with highest abundance of granule proteins. From there we believe they become engaged, so they start to release their granule content and reduce the abundance of some of the migration proteins.

the SCP data showed 7 neutrophil populations, and these matched functional states closely, showing the power of proteomics. We termed them; armed, engaged, exhausted, immunosuppressive & angiogenic, vital NETs, lytic NETs and vascular immature.

We compared tumour associated neutrophils (TANs) to the blood population, and saw they resembled mature populations. This suggested the immature neuts might not be infiltrating the tumour. We also found that despite looking more mature TANs display an intermediate mitochondrial phenotype.

The data did find clear separation for immature LDNs (CD10-), compared to the mature populations, with higher translation, DNA replication and mitochondrial related proteins being higher We also detected clear markers for CD10+ populations, which we were interested to use in SC to identify them

Between normal and low density neutrophils, there's not even a difference in the abundance of granule proteins, which we originally thought could be the cause of the difference in density. So the density changes are not proteome driven and may be related to lipids

Applying mini-bulk to blood and tumour associated neutrophils we found very interesting results: Neutrophils get classified by density, with normal (NDN) and low density (LDN) meant to be different. Our data find that at the protein level there is little difference between mature LDN and NDN

we looked at proteins important to neutrophil function and found that for many the coverage in mini-bulk and SCP was excellent. 80% of granule detected in bulk were seen in SCP, 75% of metabolic proteins and most of the adhesion receptors. However low abundance chemokine receptors were missed.

The median copy number of proteins we detect in mini-bulk rises to ~17,000 copies and when we go down to single cell this increases further to ~34,000 copies. Still some low abundance proteins in bulk are seen in single cell, as they are higher abundance in a subset of cells!

Technical details: for neutrophils 50% of the protein we detect in bulk we estimate have < 5,500 copies per cell. What happens when we go to lower cell numbers? For mini-bulk only 28% of those < 5,500 copies are detected, and only 20% when we look at SCP. So we lose low abundance proteins

Alejandro Brenes @ajbrenes.com · 10/07/2025

This is a technical feat showing the progress in sample processing an instrument sensitivity, as we estimate we inject 30pg of protein content from a single neutrophil. Yet we can detect >3,000 proteins from 500 neutrophils and >1,100 from a single GBM associated neutrophil!

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New TT wheels arrived today and it gave me an excuse to break out these bad boys. Its not just quality columns that @ionopticks.bsky.social produce, keep an eye out for their incursion into the cycling market 😁

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Alejandro Brenes @ajbrenes.com · 04/06/2025

Im a little terrified of where you live Ben 😆

Definition of the word skeet on urban dictionary where it says it means to ejaculate

Alejandro Brenes @ajbrenes.com · 08/05/2025

oh man, this gets better and better. Thermo machines now have 2 mass spectrometers inside them!