Owen Hovey

Thank you!

Can you link the original patient?

Has the ENO and ONE been compared with lower input samples 1-5ng?

Very interesting study, would have been nice to see a SP3 workflow added.

Excited to share part of my PhD work is now on bioRxiv! We show how LIN28A drives imatinib resistance in CML by reprogramming the kinome. Targeting LIN28A or downstream kinases restores drug sensitivity.

Huge thanks to my amazing collaborators!

#Proteomics #MassSpec #CML #TKIresistance #LIN28A

It’s Canadian, but the Lake Louise Tandem MS is more technological and also good if you like skiing.

Are you using SP3 or S-Trap?

See if any of the +1 match this list. If so then you have your answer where it is coming from

proteomicsresource.washington.edu/protocols05/...

What are the charge states at the end of the gradient?

Shadowing a postdoc from one of my committee members during my master's.

Universal Pretreatment Development for Low-input Proteomics Using Lauryl Maltose Neopentyl Glycol In BriefWe developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method for low-input proteomics. LMNG surfactant is added for reducing protein and peptide loss into th...

I don't have experience with EvoSep. However, they do see it at the end of the gradient in this paper in Fig 5A on Evosep. On our Neo-eclipse setup, I've only ever observed it as the dimer 1021.62 at the end of the gradient when we ramp to 95% B. www.mcponline.org/article/S153...

A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl β-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3–4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%–10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 μg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.

It comes off as a dimer at 1021.62 at the end of gradient. See supplemental figure 3 doi.org/10.1021/acs....

We’ve done a small amount of testing so far with LMNG in digestion buffer and resuspension with DDM, and we have improved recovery. DDM always comes off the end of the gradient as a dimer.

A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We pres...

Also DDM coated tubes improve phosphoproteomics pubs.acs.org/doi/10.1021/...

Universal Pretreatment Development for Low-input Proteomics Using Lauryl Maltose Neopentyl Glycol In BriefWe developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method for low-input proteomics. LMNG surfactant is added for reducing protein and peptide loss into th...

This paper compared DDM and other similar surfactants for multiple applications (phospho, AP-MS, EVs and single cell) in digestion and resuspension.

www.mcponline.org/article/S153...

Was this run on a desktop or server?

@resynbio.bsky.social has also been labeled as spam

YouTube video by Max Quant MQSS 2019 | L15: Computational PTM proteomics | Kirti Sharma

MaxQuant YouTube videos are a good starting point. There are also lots of great R scripts.

youtu.be/fdd9MPt2sAU?...

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients* Orbitrap Exploris 480 MS with FAIMS Pro provides fast, sensitive and robust profiling of proteomes when combined with Evosep One. The combination of Data Independent Acquisition (DIA) and FAIMS with s...

We do DIA without FAIMS on our Eclipse, and it hasn’t given us that much of a boost for our normal workflows. We did see a boost when exploring low inputs below 1ng. Here is a publication of the Olsen lab using FAIMS DIA.

www.mcponline.org/article/S153...

This paper from the Aebersold lab. Depending on the cell type, 0.06 (CMP) to 0.5 (GMP) R^2

Does anyone know how to extract how many MS2s contain particular contaminate fragments? (i.e. the percentage of MS2 with 136.061 m/z Adenine accounting for ppm error) Either with R, python, MSfragger, MaxQuant or Skyline is preferred. #massSOS #proteomics

I'll be back in the lab on January 7th. Can you wait until then?

We've only used Thermos barcode plates. Could you try purchasing a thermos 96-well plate and then making a copy of the barcode or transferring the barcodes to new plates? It looks like I could peel off the barcodes.

HUPO in Toronto Canada

What’s the average peptide length? What’s the cost?

FEBS Press To improve (phospho)proteome coverage, we characterized EndoPro, a proline-specific protease. The protease exhibits high specificity for cleavage C-terminal to proline and alanine and is interestingl...

Alternative enzymes to trypsin give complimentary phosphoproteome. Many amino acids are inaccessible via trypsin.

doi.org/10.1111/febs...
And

doi.org/10.1016/j.ce...

With Ryzen 7950x reaching 32 threads. A threadripper may not be needed for most, but maybe your new TimsTOF ultra will run through samples fast enough to need a threadripper.

I have not compared and don't have access to a threadripper. Though they use the same architecture, I assume IPC would be similar. The main advantage is an increased number of cores and a number of PCI lanes. The number of PCI lanes don't matter much, as we don't need them for multiple GPUs.

I use a Ryzen 2600x with a 1TB 960 Evo M.2 drive and 48 GB RAM. It finishes much faster than any of the more expensive Lab computers with Xeon processors. I keep the task manager open when running FragPipe, MQ and DIA-NN; CPU and disk speed are always limiting factors on all lab computers.

Full Mass Range ΦSDM Orbitrap Mass Spectrometry for DIA Proteome Analysis www.mcponline.org/ar...

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#proteomics #prot-paper