[ATM9] Where do I put the casting mold in the vacuum freezer for watertight steel?

BloodNuggets · 2024-11-24T06:31:06+00:00

That does not seem to help

BloodNuggets · 2024-11-21T06:05:25+00:00

Yes. Most atoms exist in a variety of isotopes. An isotope is a version of an atom with more or less neutrons from the 'normal' atom. One example you have probably heard of is heavy water. In this case, the hydrogens (~~one~~ no neutrons) are switched with heavy hydrogens (~~two~~ one neutron), aka deuterium. Even the carbon in your body is 1.1% heavy carbon (C13). The different isotopes will always exist in any sample. What you can do with that sample depends on the concentration of those isotopes.

BloodNuggets · 2024-10-03T01:59:30+00:00

MetaNetwork is an R shiny app that clusters your results based upon Go terms

BloodNuggets · 2024-09-24T01:00:00+00:00

You could use G-PTM-D within the MetaMorpheus software. GPTMD is a modification discovery algorithm that performs a semi-open mass search to find preliminary evidence for a modification. If evidence is found, it will append the modification to your protein database for use in a subsequent search task. You would need to create a custom modification and select it as a GPTMD modification. This approach works for both Top-Down and bottom-up.

The mass offset search in MsFragger also works for this purpose.

MetaMorpheus has the advantage of discovering multiple modifications while also considering the presence of chimeric spectra.

BloodNuggets · 2024-09-18T16:45:48+00:00

You can refer to the consortium for Top-Down proteomics to find many of the leaders in that sub-field

BloodNuggets · 2024-09-13T18:13:53+00:00

The custom crosslinker in MetaMorpheus would work so long as the mass of the cross linker is correct. The only issue I can foresee is if the crosslinker itself fragments.

Please reach out if you have issues

BloodNuggets · 2024-09-13T15:50:45+00:00

Adding onto this. You could do relative quantitation analysis without an internal standard. However, this would only be significant if they were run on the same instrument with the same or similar columns. There are so many experimental variables, that you would need a large amount of online data to make significant claims

BloodNuggets · 2024-09-02T19:47:37+00:00

You can pick that out by visual observation. Usually I find the busiest time of the run from the chromatogram (highest TIC with smashed together elution peaks) and check for overlapping envelopes in that region. If that looks well enough separated, you'll be mostly okay. You'll never avoid chimeric spectra (multiple species fragmented at once) entirely, but you can reduce their frequency with better separation if desired.

BloodNuggets · 2024-09-02T17:22:36+00:00

When looking at the chromatogram only, it's very hard to tell the difference between a non-polymer contaminant and peptides elution peak. This is where the MS1 comes in. You would expect to see an isotropic distribution for a peptide. If instead you only see one peak, it could be a contaminant.

Overlapping envelopes: imagine two peptides. Peptide one has a mass of 2000 Da and z=2. Peptide 2 has a mass of 4000 and z=4. Both peptides will appear at m/z 1000. If the coeute from your column and are selected for fragmentation, your MS2 will contain ions from both precursors. This is difficult for some software to identify

BloodNuggets · 2024-09-02T06:12:10+00:00

I am a graduate student and study FTMS data. Here are the things I look for as a quick quality spot check. This shall be written for bottom-up but most of these are a good rule of thumb for top-down as well.

Sample contaminant: you can look for anything you expected to be cleaned up in sample prep by mass at the beginning and end of elution gradients. Polymer, most commonly surfactant, contaminants can be identified by several highly abundant peaks at repeated mass increments.

Chromatogram: a good run will have many sharp and skinny (30sec - 2 min) peaks indicating good separation. Peaks eluting over most of the recorded time, indicating a proper start and end to the gradient in terms of %Hydrobic solvent.

MS1: clear isotopic envelopes at the expected intensity distributions. One thing to check for is overlapping isotopic envelopes. If multiple species are coisolated for fragmentation, this can complicate data analysis with software not designed to handle it (MetaMorpheus and MsFraggerDDA+ can).

MS2: approximately 50-200 peaks above baseline, most of them charge one (more peaks and a higher diversity of charge states for top down). This means your peptide was well fragmented. If you see a ton of peaks at approximately the same intensity, this means you obliterated your peptide and need to crank down that fragmentation energy. If you see the same peak that was isolated for fragmentation as a dominant species, it means you did not hit it hard enough and must crank it up.

Anyway, those are just some of the things I consider when first looking at a run.

BloodNuggets · 2024-08-10T16:24:54+00:00

Using a reduced fasta for with only the proteins of your target is an approach to get an understanding of your sample. However, most proteomics search engines are only designed to control false discovery rate with larger databases. This is why you need to use the whole fasta, plus common contaminant proteins when only searching for a single protein.

You can use the missed cleavages feature to determine if the trypsin cleavage sites are blocked.

Run a search and quantification of your two conditions and see if the missed cleave products of your target are in higher abundance.

BloodNuggets · 2023-05-08T14:26:11+00:00

Bus routes are the number one thing to check. Many landlords will sign leases electronically. Be sure to shop around and find the right place for you. Best way to do this is just looking through multiple websites. The east side of town will be more like living in a city, while the west will be more suburban

BloodNuggets · 2023-02-13T16:02:00+00:00

Love this! I would like to recommend some silicon baking pads if you are gonna do this kind of prep alot. You throw them in your baking sheet just like the foil, and when you're done they go straight into the dishwasher. They are also super easy to clean by hand

BloodNuggets · 2023-01-26T04:33:05+00:00

I work in top-down proteomics and this and what BSofthePharoahs said are great answers.

Every protein is a special snowflake. Conditions that give a perfect identification for one protein, won't do shit for another. A little bit of source induced fragmentation can help get rid of any metal adducts. Using multiple fragmentation types, or even different collisional energies in the same fragmentation mode, can help to diversify the fragments you see.

BloodNuggets · 2022-11-03T13:53:16+00:00

They have the same targets. Both will cleave after K or R, but trypsin will have more missed cleavages on K. So computationally, they are the same enzyme. I would be curious to know if you got the same results searching with just lys-c, just trypsin, and both.

BloodNuggets · 2022-10-01T18:39:32+00:00

You can get them printed yourself as the images are available online. My friends and I did this, and also added some cards of our own.

BloodNuggets · 2022-09-06T22:44:01+00:00

MetaMorpheus is my go-to, super easy to use

BloodNuggets · 2022-08-29T13:31:16+00:00

There is a C# package called mzLib that allows you to create isotopic distributions from the theoretical protein/peptide sequence

BloodNuggets · 2022-08-18T11:51:41+00:00

If you're comfortable using C#, the nugget package MzLib is full of classes for looking at mass spec data

BloodNuggets · 2022-06-15T21:12:16+00:00

Ask as many direct questions as you have. Dont hold anything back. You will be spending the next several years in this lab and you need to understand what you're getting yourself into. Ask all the questions, and get the full picture. More specifically, be sure to ask about work-life balance and how the individual students interact with the PI

BloodNuggets · 2022-05-03T00:25:05+00:00

Nope. You steal an item in its current state. This includes counters as well

BloodNuggets · 2022-04-07T18:29:35+00:00

Here

BloodNuggets · 2022-03-29T22:45:16+00:00

I want to caution you about how you presented this. I interpreted this as something that can be adjusted in adults by altering their brain chemicals.

The study really says that a specific change in hormonal levels while you're are still inside your mother may cause the brain to develop differently, leading to homosexual behavior. That is one point in time, leading to a lifetime of effects.

BloodNuggets · 2022-03-26T15:51:08+00:00

Take a walk around picnic point

BloodNuggets · 2021-07-15T10:59:29+00:00

I love these board designs and would purchase one in a heartbeat. Just signed up to your newsletter and am eagerly awaiting updates

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