Finalize Protein Extraction Protocol

[sr320]

@Ellior2 is getting a markdown version of the protein extraction protocol up at https://github.com/sr320/LabDocs/tree/master/protocols

Once a draft is up lets make sure it is clear to all and we have necessary reagents.

[Ellior2]

https://github.com/sr320/LabDocs/blob/master/protocols/ProteinprepforMSMS.md

[sr320]

Nice! On Sat, Nov 5, 2016 at 12:33 PM Rhonda Elliott notifications@github.com wrote:

https://github.com/sr320/LabDocs/blob/master/protocols/ProteinprepforMSMS.md

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/sr320/LabDocs/issues/333#issuecomment-258635418, or mute the thread https://github.com/notifications/unsubscribe-auth/AEPHtzd8mY-7sIGn76KkVFdZBfaPTxETks5q7NnngaJpZM4KqWZe .

[sr320]

I have started making a list at the top of the protocol re reagents / supplies.. https://github.com/sr320/LabDocs/blob/master/protocols/ProteinprepforMSMS.md Please add to, and confirm

[Ellior2]

Added some more things to the list.

Clarification question as I can't quite remember:

You can do steps 1-8 from the mini-trypsin digestion before knowing how much protein you have per sample? Then once you quantify the proteins using the BCA assay, you can continue with steps 9-16?

[sr320]

@emmats can you clarify

[Ellior2]

Also can we get clarification for Step 1 in Sonication:

It states: Add 100ul 6M urea in 50 mM NH4HCO3 (ammonium bicarbonate).

But really we... Smash frozen biological material with pestle in centrifuge tube. Then add (volume?) 50 mM NH4HCO3 (ammonium bicarbonate). Then add 100ul 6M urea.

Is this correct?

[emmats]

First question: Yes, you do not need exactly 100 ug of protein until you start adding enzymes. The other reagent amounts are based on your sample volume. The amount of enzyme added depends on the amount of protein in your sample. You can always do your BCA assay early and do the entire thing on 100 ug of sample.

Second question: Not correct. You make a solution of 50 mM bicarb + 6M urea. Here is the link to my notebook entry for digestion: https://www.evernote.com/l/APLBpNYN-VlFvbPyRfi7DbOQX3q2u6s2mbQ

[Ellior2]

Thank you @emmats . As far as technical replication goes- we decided to just do this on one sample to verify that there is no variability in our techniques right? So on the first day, for example, I could make 3 replicates of sample #1 starting from sonication (1A, 1B, and 1C)? Is it necessary to do this for each of the 3 extraction sessions?

[emmats]

You are referring to technical replication of extractions? And you would digest 1A with the first round, 1B with the second, and 1C with the third? Did you look at the text book I forwarded to you? Is that what they suggest?

[Ellior2]

We decided to cut down the number of samples so I might even be able to do it in one session. In which case, subdividing one of our samples into 3 replicates for digestion would allow us to see potential variability in the digestion process. After desalting, we could also create 3 replicate vials from that same sample to test variability in MSMS. If there is variation in either step, we should be able to tell whether it was during the digestion/desalting or the MSMS. It should only create 2 extra samples for digestion and 4 extra for MSMS.

I'm still confused about the 50mM bicarb + 6M urea. How specifically would I make that solution? I use 500ul 6M urea and add it to how much 50mM bicarb?

[sr320]

I suggest adding the presumed method to making everything at the beginning of the protocol - then we will review all.

[emmats]

For the bicarb, you make 50 mM NH4HCO3. You then add enough urea to equal 6M.

[kubu4]

@emmats - Can you clarify this a bit more? So, what is your final NH4HCO3 concentration???!!

[kubu4]

Oh, wait. I hit enter too soon. I see...

The solution (homogenization buffer) should be 50mM NH4HCO3, 6M Urea.

The confusion stemmed from the wording about in your notebook "Added 500 µl 6M urea in 50 mM bicarb". It sounded like the instructions were to add a solution of urea to a solution of 50mM NH4HCO3.

Thanks! I think we are clear, now.

[emmats]

You got it @kubu4 :)

[Ellior2]

I updated the protocol with how I think the reagents should be made... please review... https://github.com/sr320/LabDocs/blob/master/protocols/ProteinprepforMSMS.md

[Ellior2]

How to make reagents via @emmats Lab:

1.5M tris HCl - 1.8g in 5 mL 200 mM DTT - 154.25 mg in 5 mL 25 mM NH4HCO3 - 0.1976 g in 100 mL 50 mM NH4HCO3 - 0.395 g in 100 mL 6M urea - 1.81 g in 5 mL 200 mM TCEP - 286.65 mg in 5 mL 20 mM IAA - 184.96 mg in 5 mL

All of these match up with my calculations except the Tris-HCl. What molecular weight do you have Emma ? I am calculating to add 1.182g in 5ml

Can you verify my method for making the ACN solvents as well?

Thank you

[kubu4]

@Ellior2 - In regards to the Tris solution, you'd be better off using Tris base (it's basic, instead of acidic - the protocol indicates that the solution should be pH = 8.8). This could explain the difference in calculations you.

Also, the methodology you have written up for making solutions is incorrect. Here's an example:

Dissolve 36.036g Urea into the same 100ml solution

This is wrong because adding the solute to the solvent will increase the volume of your solution. Thus, you will end up with a solution volume greater than 100mL and you will not have the desired final concentration.

For making solutions, always dissolve the solute in the smallest possible volume of solvent. Once the solute has fully dissolved, bring the solution to your desired final volume.

[Ellior2]

Yeah, I was thinking about that. I guess I am just unsure how you would "top off" to exactly 5.00ml. I need some guidance on how to make such exact small volumes of solutions.

[kubu4]

Options:

• Make larger volume that can be measured more easily (e.g. 50mL)
• Use a measuring device capable of measuring a small volume accurately (e.g. serological pipet)

[emmats]

@kubu4 is right, of course, but you can ignore him @Ellior2 for the ~6M of urea. It doesn't need to be so precise so you can just use the numbers I gave you. Unless you want to make things slightly more difficult for yourself!

[Ellior2]

Ok. Are you using Tris or Tris-HCl since the pH has to be 8.8? And what is the molecular weight you use to calculate the 1.8g Tris in 5ml?

Also, can you check my ACN solvent calculations?

Thank you @emmats

[emmats]

My notes say Tris HCl. I will follow up with Brook to make sure since there is some confusion. I do not have MW numbers, I just copied what was written on our bottle of Tris. I will double check 1.8 g when I am in the lab tomorrow.

For the ACN solvents the 60% looks good, but the numbers are off a little bit for the 5% (I think you are making a 4.7% ACN and .9% TFA). Note that when you are making these solvents you should not use plastic. Use glass graduated cylinders and for pipetting small amounts (like TFA), don't pipette directly from the bottle (i.e. plastic should never touch your stock reagents). The reagents can erode plastic a little and the plastic particles will be detected in the mass spec.

[Ellior2]

Hmmm... I'm not sure where I went wrong. I calculated the two solvents using the same logic.

For the protein quantification part, what concentration of 22 ul of ammonium bicarb are we adding to our 11ul of sample to dilute the urea? 50mM?

Sorry to hammer you with questions.

[emmats]

First, updates from Brook: Yes, we use Tris-HCl to make our Tris Apparently I can't read and it is 1.18 g in 5 mL for the tris

Yes! You are adding the 50 mM.

I don't mind answering questions. This is better than something going wrong and trying to figure out what it is after wasted time and sample.

[emmats]

P.S. generally, don't put plastic pipette tips into concentrated acid.

[Ellior2]

Do we need special pipette tips? How do you do it?

[emmats]

Not special tips, just use glass as much as possible and pour a small amount into a beaker if you need to use a pipette. That way any dissolved plastic won't accumulate in your stock solution.

[emmats]

Brook also wanted me to remind you to make sure you don't overheat the samples during sonication.

[Ellior2]

Ok, I'm just going to follow the protocol but I'll be cautious of that. Thank you. I'm going to make some of the reagents tomorrow, the ones that we have chemicals for and won't expire. For the tris-hcl with pH8.8, what should I add if the solution is too acidic? Too basic? And can that be stored on the benchtop?

[sr320]

@kubu4 should be able to address any questions re making solutions in lab tomorrow, I will be available in lab Thursday AM.

Importantly, document everything you do... so someone could reproduce....

On Tue, Nov 15, 2016 at 9:08 PM Rhonda Elliott notifications@github.com wrote:

Ok, I'm just going to follow the protocol but I'll be cautious of that. Thank you. I'm going to make some of the reagents tomorrow, the ones that we have chemicals for and won't expire. For the tris-hcl with pH8.8, what should I add if the solution is too acidic? Too basic? And can that be stored on the benchtop?

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[emmats]

@Ellior2 , yes it can be stored on the benchtop.

I would add an acid or a base :) Probably relatively concentrated NaOH or HCl. There should be some near the pH meter, I think. @kubu4 can probably be more specific.

[Ellior2]

Detailed sonication steps for review @emmats @sr320 . I plan to do this on Monday if @laurahspencer or @yaaminiv want to help!

We will need to get dry-ice from the Health Sciences J-wing in the morning. What information do I need to get the dry-ice (i..e. Lab code?)

On Wednesday I plan to do the BCA assay which will set us up nicely for the mini-trypsin digestion starting the 28th.

[emmats]

You shouldn't need tongs for dry ice because they have a scoop. You will need the SAFS box number and a Roberts Lab budget number.

Yes, save all sample you don't immediately use at -80.

I usually take a ~500 mL beaker, fill about halfway with EtOH and then add 2-3 pieces of dry ice at a time. Eventually you will see that the dry ice doesn't dissolve fast and that means the EtOH is nice and cold.

At the end of sonicating, I would aliquot the volume you will use for protein quantification so you don't have to thaw the entire sample when you quant it.

I can come by the morning of the 28th to see how things are going. I can stay until about 11:30, if necessary.

[Ellior2]

Ok, so aliquot 11ul of each sample into a new tube to freeze for the protein quantification? But then when I start the mini-trypsin, I will only have 89ul. Do I add 11ul of something to get back to 100ul and then determine in step 9 how much of this sample I will need based off of my "new" protein concentration, since I just diluted it?

[Ellior2]

Can I just take like 150ul of the supernatant instead of 100ul after the centrifuge step. That way I have enough for the protein quantification AND the digestion?

After sonication, for each sample I could aliquot 11ul for the quantification and 100ul for the digestion and freeze those.

[kubu4]

Heads up on a few things:

1. Most people will be gone from lab on Monday (big sampling at Manchester), so this might not be the best day to run through the proceure?
2. You're methodology for making the homogenization buffer is incorrect. If your final desired volume is 20mL, then you can't put 20mL of water in a conical tube and add solute to it. Your final volume will not be 20mL. Don't make the solution this way. It's not right and you don't want to establish any bad habits...
3. Make sure to use a plastic container for your dry ice/EtOH bath!
4. Are you processing actual samples? I'd highly recommend going through the protocol with some "practice" samples that are not critical for your experiment if something goes wrong. The protocol is still not clearly written in a number of places, so it wouldn't surprise me if you encounter some unexpected difficulties along the way that could ruin your samples.
5. Have you compared the Roberts Lab sonicator to the one that Emma uses in her preps? Are they comparable? Will 10s of sonication be sufficient with our sonicator? Will it be too much?
6. I don't know if we have any clean plastic pestles. You should definitely check this before beginning...

[emmats]

@Ellior2 you can sonicate as much of the supernatant as you want.

Hm, I always use a regular beaker for the etoh + dry ice. I haven't had any problems yet, @kubu4 !

[Ellior2]

Thanks @kubu4 ! I did facetime with Emma this morning and showed her our sonicator, I think it'll work just fine. I tried sonicating a 100ul water sample in a tube for practice today, and it definitely heated up when sonicating for the full 10 seconds. We decided to cut it down to 5s and since we are doing it three times, we should be good.

I do have duplicate samples still in the -80C for this experiment and we should have leftover sample too so I am just going to go for it. Otherwise, I'm never going to get these done if I want to run "practice samples" on the MSMS before actually starting to digest mine.

Here is updated sonication steps

I'm breaking the whole process down into different steps and I won't pull anything out of the -80C until I know EXACTLY what I am getting myself into that day.

[kubu4]

Excellent!

[Ellior2]

@kubu4 can you provide me with SAFS box number and Roberts Lab budget number to get dry ice tomorrow? I went with Steven and Holly to a place near the Bagley building where I can get dry ice too. I assume I'll need the same information there.

[Ellior2]

Nevermind let me guess... 355020 and 61-0221

[kubu4]

Box number is correct. I think that budget number is good, too.

[Ellior2]

Also @kubu4 I can't see where the BCA assay kit was put. I see that it came in under the purchasing log, but I don't see it listed on Mychem so I don't know where you put it. I am just hoping to see if there are directions that come along with it so I can be prepared for the next step. Thanks

[kubu4]

It's on the shelf with the rest of the kits in 213.

[Ellior2]

@emmats

For the protein quantification, do you do three replicates for each sample, and three replicates for each standard?

There is also supposed to be a blank with just Lysis buffer which I didn't see in your table.

[emmats]

Yes

[Ellior2]

@laurahspencer and I will be starting on the BCA assay procedure tomorrow morning (Wednesday)

[Ellior2]

@emmats I've got a few questions for the digestion process I hope to do Monday:

1) For 5 of my samples, I have less than 1ug/1ul of protein, which means that I will have less than 100ug of protein for steps 1-8. For the remaining samples in which I had more than 1ug/ul I am diluting so I have exactly 100ug of protein in the 100ul required starting volume. So at Step 9, can I continue when some of my samples have less than 100ug? Do I just adjust the amount of LysC and trypsin?

2) Is the process for adding Lys-C the same as the Trypsin process that you described in more detail since they are both (1:30 enzyme:protein).

From your notes: Prepare the number of trypsin bottles needed (you will want 5 µg of trypsin for each sample, or 1 µg trypsin: 20 µg protein). Add 20 µl water or trypsin buffer to each bottle of trypsin and vortex lightly. Aliquot 5 µl of trypsin to each sample for 100 µg of sample (1:30 enzyme:protein).

But is it a 1:30 ratio or a 1:20?

3) For the trypsin, I calculated how much I will need based off of the amount of protein in my samples and it looks like I will need 105.4ug so we are 5.4ug short. Can we borrow some from your lab and order more? If I am correct in assuming you add Lys-C in the same concentrations as you would the trypsin, then I would also need 105.4ug, again possibly 5.4ug short. We also haven't received our trypsin and don't know if we will by Monday. It's the last thing we need.

Thank you!

[Ellior2]

@sr320 @kubu4 Do we have a 37C incubator in our lab for Step 4?

@emmats Assuming I have enough enzyme to go through with this Monday, would I be able to use your cold speed vac on Tuesday?