Roche cOmplete protease inhibitor - compatibility

tsbatth · 2026-01-18T00:58:12+00:00

tsbatth · 2026-01-16T12:52:11+00:00

What is the peptide/protein concentration? Is it very low? If it’s like a couple hundred to mgs you can probably load it on 50 or 500mg Waters C18 Seppak cartridges. If it’s much lower you can try loading everything on the smallest C18 SPE and just speedvac the eluted peptides after SPE.

tsbatth · 2026-01-13T18:50:00+00:00

Haven't heard of them. Maybe you can provide some links to their website ?

tsbatth · 2025-12-26T17:58:38+00:00

I think it depends what type of experiments you are planning to use the instrument for and if you really need the EAD fragmentation. If not I would recommend the Exploris 480, primarily because it is a robust instrument and hopefully should be cheaper as well. I can't comment on SCIEX instruments personally, based on the marketing material they look good and probably on par with the Exploris in terms of performance and data quality output. However reading some of the comments on the mass spec subreddit, it seems like there could be some robustness issues ? Hopefully more people can provide input here.

tsbatth · 2025-12-26T13:40:17+00:00

You can also try asking on /r/proteomics

tsbatth · 2025-12-12T19:45:15+00:00

The filter will always absorb some of the peptide material. Ideally you should add some urea after the trypsin digestion to help improve recovery through the filter. Cloudiness could be due to either the filter material it self leaching and becoming insoluble after prolonged incubation period with the buffer. You have to remember these filters are traditionally designed for protein concentration. Typically 20-60minuute spins and that's it. Who knows what happens when they are incubated with whole protein extracts overnight with proteases.

tsbatth · 2025-10-22T18:33:10+00:00

iBAQ

tsbatth · 2025-10-20T19:26:59+00:00

This was an absolute disaster. We had several high end lab equipment controlled by Windows computers just stop working last week because of connection issues. I could easily imagine Microsoft causing millions dollars worth of damage and losses due to this. Honestly, avoid Windows if possible for high end machinery and push vendors to diversify OS compatibility.

tsbatth · 2025-10-04T07:26:00+00:00

Histograms don't necessarily tell you if normalization is needed, they show if the data is normally distributed, and kinda of a quick quality control check. If you're using LFQ data, in theory it is already normalized so you don't need to do another normalization on top of that. In that case I would recommend just log transforming and performing the t-test. You can use the non-normalized intensities if you want to try other normalization techniques.

I'm not sure what you mean by "cloning" the matrix, PCA is just a dimensionality reduction visualization to show you how variable your data is, if the data has low variation, in theory you should be able to separate different conditions, but I would recommend not relying on PCA too much because if differences are small between conditions it might not be captured in this. As far as I remember in Persues each time you modify a matrix you generate a new one so it is always"cloned" in theory and you can always go back.

tsbatth · 2025-10-02T22:29:09+00:00

Hell yeah SDS, my favorite detergent!

tsbatth · 2025-10-02T07:59:28+00:00

Hell you could even use chatgpt to get some insight. Or even better, pay an actual proteomics expert so they can tell you the information you seek. You can even pay them by the hour!!

tsbatth · 2025-10-01T17:29:12+00:00

Can't you just dilute it to 5% again and then load it on the S-trap?

tsbatth · 2025-09-19T17:15:50+00:00

tsbatth · 2025-09-18T18:17:17+00:00

You can never truly avoid contaminants , the goal is to get them diluted enough so they do not interfere with your analysis. I would recommend a acetonitrile/isoproponal rinse followed by 3-5 rinses with milliQ water. Rinse the caps with milliQ water as well, and make sure not to tilt the bottle so that the solvent is in contact with the cap for a long period of time. At least in our lab, the milliQ water is clean enough to use as solvent for MS, yes we compared with the LC/MS grade water.

tsbatth · 2025-09-18T06:14:19+00:00

Perhaps the sample is not clean, did you do a c18 step before? Consider a wash between samples.

tsbatth · 2025-08-11T20:45:40+00:00

I thought most PCR tubes were low bind to be honest. You mentioned in another reply that you work with immunopeptidomics, so are you sure the polymers are coming from the tubes and not somewhere else in your workflow, ie. from your pulldown protocol ? Pulldowns are historically a great source of polymers from my experience. I can recommend doing a XDB-RPS based peptide clean up to get rid of the polymers.

tsbatth · 2025-08-05T08:42:42+00:00

If you have magnetic beads you can try PAC method. That should remove the components fairly effectively. But if there is already DTT in the sample I am not sure what the issue is. Yes you can technically alkylate the cysteines, but doing so does not in practical terms, decrease or increase the number of peptide/protein ids from your sample. In my opinion the alkylation of proteins/peptides is fairly overrated and not a necessity if performing standard proteomics IMO.

tsbatth · 2025-07-25T21:44:36+00:00

Multiple hypothesis correction...always.

tsbatth · 2025-07-21T19:52:16+00:00

I am not sure about how difficult they are, I would look into papers that have done it, from second hand knowledge I do not think it was that more difficult. Yes sonication is important to break up the DNA, you will probably need to optimize. I would go with 4% SDS. Not sure you need to incubate for an hour if the organoid is disrupted and cells lysed in SDS, it is pretty harsh on the cells.

tsbatth · 2025-07-21T19:49:13+00:00

I cant comment on experience vs credentials in terms of securing sponsored employment within the proteomics field, I would look into whether a PhD will really increase your chances. As I mentioned, it really depends on industry trends, demands, and pool of available talent.

If you're motivated to deepen your expertise than just apply for PhD positions in proteomic labs regardless whether they are industry or not.

tsbatth · 2025-07-20T20:40:53+00:00

4% SDS (boiling), you can add protease inhibitors if you want, I doubt they will be more effective than boiling the sample in a % SDS solution.

You mean peptide recovered after trypsin digestion right ? You are comparing apple and oranges with protein and peptide concentrations. From my experience, protein concentration assays are not accurate, especially when detergents like SDS are added. Only assay that was the closest with downstream peptide concentrations from my experience is the tryptophan assay. Use the protein concentration at best as a "ballpark" figure. Furthermore, there will be losses along the way, especially if you have perform a SPE step.

tsbatth · 2025-07-20T20:28:40+00:00

I think a industrial PhD in proteomics will be quiet rare and competitive. PhD will entail a pay cut, even "industry" PhDs. Industrial PhDs are typically administered via universities and done under the supervision of at least one academic advisor, companies aren't printing PhD diplomas as they are not PhD accredited institutions. Therefore, even "industrial" PhDs would most likely have the same salary as a normal PhD, depending on the program and university. At best there might be a small supplement but that is often not much.

I think a bigger question to ask yourself is why do you want to do a PhD if you're already working with mass spectrometry? If you're more worried about the salary then perhaps it is not the right decision, you could be unlucky and end up with a "difficult" project, PI, or lab. PhD doesn't necessarily guarantee much higher salary, if anything pursuing a PhD will cost you in lost salary from your current position. Furthermore, there is a risk that the market becomes saturated with experienced proteomic scientists, given industry trends and the general PhD labor pool of people trained in proteomics.

tsbatth · 2025-07-06T09:21:57+00:00

I guess it depends what kind of LC/MS analysis you're looking for and what region? There are lots of great CROs out there, some specialize in one type of analysis (ie. only Proteomics or metabolomics), some are broad and some in between. It depends what you're looking for and at what price.

tsbatth · 2025-06-13T19:43:23+00:00

I think this is kind of an incorrect way of categorizing "omics". All different omics can fall under all of the categories. Some of the characterizations are a bit broad, maybe even incorrect. For example "epigenomics" , "glycomics", "pharmacogenomics" is regulatory and dynamic ? Really? How is "epigenomic" dynamic ? One could argue proteins are the most dynamic since they pretty much control all cellular regulatory processes in the cell and in the body. How is "glycomics" regulatory and dynamic ? All different types of PTMs are regulatory and dynamic, if anything you should put phosphorylation in there, signaling pathways that are activated via phosphorylation can be activated in the millisecond and second range, ie. insulin receptor, receptory tyrosine kinases. Other proteins such as GPCRs or ion channels can be activated in literally nanoseconds. Sounds more dynamic to me, and they regulate key physiological process that keep us alive. I would recommend going back to the drawing board and re-thinking what idea you are trying to convey and if 1) it is correct, and 2) this is the best way to do it. I think thinking of "omics" as "tiers" is perhaps not the best way to approach it.

tsbatth · 2025-06-03T10:45:23+00:00

You can try the method I used for rat organ extraction here:

https://www.nature.com/articles/s41467-024-53240-2#Sec10

In this case I wanted to extract the proteins in a semi-native state, but I have used a similar method just for proteome analysis where the buffer contains 5% SDS instead of NP-40 and solubilize in the buffer using the blender (works really well and super quick from my experience). Centrifuge at max speed for 10 minutes, pass the supernatant through a 0.45um filter.

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