- by /u/FactorAgreeable7518I am currently optimizing a workflow in which I aim to begin with 10% ACN in biofluid (samples having 10ug of protein in BCA estimation) on a 10 kDa filter, collect the filtrate (degradome), and then resuspend the retentate (the top part on 10KDa) in 8M urea buffer to proceed with the standard proteomics preparation (reduction, alkylation, trypsin digestion, and quenching). After trypsin quenching in the retentate , I aim to mix the filtrate (degradome where I assume to have endogeneously processed peptides) with trypsin digested peptides and run them in LCMS (DDA). The overall objective is to identify microbial […]
- by /u/Expensive-Painter-18We analysed cell lysate from HEK 293 cell line on Waters Xevo G2 system in nanomode and same sample was also provided to Sciex facility where equal load was tested on Zeno ToF. The difference in number of IDs is huge! Xevo with 150 mins run time could barely ID 1500 proteins while Zeno ToF with 30 mins run time easily churned out 3500 protein IDs. I know Xevo is an older model but even QE Orbi which is released in almost same year as that Xevo will easily outperform it. Where do Waters systems suck? I see good MS1 […]
- by /u/kinder_brzI’m experienced in basic data analysis but new to clinical omics integration — especially linking omics data with patient outcomes, treatment groups, and survival/time-to-event statistics (Cox models, hazard ratios, etc.). Could you recommend any books, GitHub repositories, YouTube tutorials, or online courses that teach how to integrate proteomics data with clinical data and perform downstream statistical and bioinformatics analyses? Preferably R-based resources, but Python ones are also welcome. Thanks in advance! submitted by /u/kinder_brz [link] [comments]
- by /u/MagnusLocoHi, I would like to know how can (if I can) compare the proteome of species A, B and C (same genus), given that they were identified and quantified individually. I ran an Orthogroups analysis to find the proteins orthologs. Do you think I could draw "direct" comparisons, like "protein X has 2 log fold change in species A compared to B" ? submitted by /u/MagnusLoco [link] [comments]
- by /u/CoolBanana0Hi all, Looking for some hardware/feasibility advice. Our institute recently aquired a new Thermo Vanqish (flex, not neo) and Orbitrap Exploris 120 with the hope of doing proteomics. I've spent most of my PhD making proteomics probes and doing in gel flourescence but requiring collaborators to aquire proteomics data for us but we are now looking to move things in house. Unfortunately we do not have the budget/expertise for setting up a full proteomics lab. Looking for some advice to see if the equipement we have is capable enough to get some meaningful data. From what I can see: The […]
- by /u/RumbleStrut84I have always been collecting MS2 of digests before TMT labeling using an orbitrap-ion trap MS2 method with FAIMS on a tribrid mass spec. I have a very small coIP sample and I need to do a simple ID and have been asking around what methid people prefer. the couple of people I spoke with seem to prefer an OT-OT method without FAIMS, but I get far fewer IDs with a HeLa digest with such a method. I understand that the MS2 spectra would be higher resolution, but if you want more depth is there a strong reason why people […]
- by /u/XierraxAlthough this question applies to any kind of high dimensional data, I am the most familiar with proteomics and hope this is a good place to ask. Especially in a group that lacks biological expertise, once we have our set of differentially expressed proteins in healthy and diseased samples, how can we ensure that our interpretation of the results is sound? Sometimes even downstream gene ontology or pathway analysis can give vague results that can be spinned in many ways (e.g. immune response can be detrimental to a tumor or beneficial). How to avoid the trap of red herrings? As […]
- by /u/Disastrous_Bad3802I'm having trouble resolving data concerning two unique peptides that have the same KxGG and carbamidomethyl modification, but one has an additional oxidation modification (see attached). Peptides have already been filters using Percolator PEP and q-values. I have multiple biological replicate samples, and some samples show a signal for either the oxidized peptide, the non-oxidized peptide, or for both. If this is the case, should I collapse the signals from both peptides into one by calculating the median signal value? There are also unique peptides identified that have the same KxGG modification, but different peptide sequences due to alternate chymotrypsin […]
- by /u/bluebottl3especially for targeted analysis since it can do 100+ proteins at abs quant? is the fusion protein the main limitation? Thought that with the advances in AI those can be solved for faster? submitted by /u/bluebottl3 [link] [comments]
- by /u/ApexDrifter_07Hi everyone I’m working on proteome-expression analysis for lung cancers (LUAD + LUSC) and trying to download the right files from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) / Proteomic Data Commons (PDC) portal. I’ve found the right study pages (e.g., for LUAD), but I’m stuck on: – identifying the correct processed proteome expression matrix (versus raw spectra etc), – finding the correct metadata/clinical file that aligns with it. If anyone has done this and can share exactly which file names (or a link) they downloaded for LUAD and LUSC, that would be super helpful. Also if you have any […]
- by /u/RendertheFatCapHey all, another question for the experts. As my lab is doing more proteomics, we're expanding to increase proteomic depth and coverage with high pH fractionation/concatenation. Is the typical Top3 method for protein LFQ quantification still valid under a fractionation/concatenation scenario? Or does the variable peptide recovery across 2 dimensions + any drying of fractions mean LFQ isn't possible? Can it be done, as long as each fraction is normalized to a similar injection volume/concentration? I've seen papers where people use heavy peptides for absolute quantification in plasma and a couple where the 2D fractionation/concatenation is used for LFQ with […]
- by /u/OmicsAndOmHey everyone, I’m trying to break apart very stable protein aggregates in preparation for mass spectrometry analysis. My goal right now is just to confirm that I’m getting enough protein and that the aggregates are actually being solubilized before moving on to MS. Following a couple of papers, I’ve been treating the aggregates with 90–100% formic acid for 1 hour at 37°C, then using a SpeedVac at room temperature for ~1 hour to dry and pellet the denatured proteins. The issue I’m running into is that when I try to measure protein concentration using a BCA assay, I don’t detect […]
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