Dear ABRF, I am having Procise detectors fail at an alarming rate, is anyone aware of someone who can completely rebuild these? I have a stack of dead ones and am very interested in any help or ideas that someone might be able to provide to keep this service up. Thank you, John Schulze Molecular Structure Facility University of California, Davis 530.752.7327 http://msf.ucdavis.edu/
Dissolved gasses in the solvents will degas and form bubbles when they enter the low-pressure region after the column. The bubbles will disturb the light in the flow cell of the detector and cause the output to be erratic. This is especially problematic on high pressure mixing HPLC systems which typically do not have in-line degassers or helium sparging of the solvents. You should do two things, degas your solvents by vacuum filtration, or vacuum and sonication. Second you can add a smaller inner diameter restrictor line on the outlet side of the detector to maintain a higher pressure on the solvent all the way through the detector. Be careful not to use too small a restrictor for your flow rate and solvent viscosity which could generate too much back pressure causing the flow cell to leak or break. For example, using a length of tubing on the detector outlet with inner diameter of 0.005 inches will generate several hundred PSI depending on flow rate and prevent degassing in the flow cell. The Waters detectors I've used can take up to 1000psi. You also mention that the sample energy and reference energy readouts are fluctuating. If you do have solvent flow going through both the sample and reference cells, then the above is the possible problem. I typically never used the reference cell so if both energy readouts are fluctuating with no flow going through the reference cell, then it's likely your lamp is going bad and not much light is getting through the flow cell to the photodiode resulting in lots of noise. The noise will become greater when there is absorbing material flowing through the flow cell. For example, even with a new lamp, if your peak contains a large amount of absorbing material that causes the detector response to go off scale, that can cause erratic output during the center of the peak elution because the analyte is absorbing nearly all the light and little energy reaches the photodiode resulting in erratic output by the detector. If you combine this situation with a weak/bad lamp this will exacerbate the problem. All of this troubleshooting assumes your pumps are working properly. Best regards, Brian
________________________________ From: ABRF Discussion Forum <email obscured>> on behalf of Anderson, David Eric (NIH/NIDDK) [E] <email obscured>> Sent: Thursday, January 14, 2021 5:01 AM To: <email obscured> <email obscured>> Subject: Re: [ABRF Discussion Forum] Shimadzu HPLC issue CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email system. Whether the sender is known or not known, hover over any links before clicking and use caution opening attachments. I am not familiar with this unit but you have 3 different things to isolate. You may have bubbles in the detector. refreshing the cell or line with water than lots of methanol then water (all degassed/sparged) would treat that. It doesn't sound like an actual peak shape problem from fluid connections but a bad connection can help put in bubbles etc. Finally you can check electrical connections including the outlet connections if you use those. This sounds like bubbles or noise. Hopefully someone with knowledge of this actual unit will respond later in the day. All the best, Eric D. Eric Anderson Mass Spectrometry Facility, NIDDK NIH Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA (301)-496-7546 ________________________________________ From: OKARVI, SUBHANI MOHAMMAD <email obscured>> Sent: 14 January 2021 02:28 To: <email obscured> Subject: Re: [ABRF Discussion Forum] Shimadzu HPLC issue Hi there, I am having some peak shape issues, with the Shimadzu HPLC system SPD-10A VP UV detector. S.E. and R.E. values (mv) are keep changing and blinking on the system monitor. The D2 lamp used time is 1192 hours. Any suggestion regarding this issue will be appreciated. Subi โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F2Qo0Gfkx0SmFvjyOEjTy79&data=04%7C01%7Cbhampton%40som.umaryland.edu%7Cb57831c773324018bb9508d8b8735fd4%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637462153217869399%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=erURhgqfZPh6dqtv2r6YK%2BsAhJfIUpqSJQZHQMXhhDk%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F5CYPGJuUdZkDPDTIHtOTv5&data=04%7C01%7Cbhampton%40som.umaryland.edu%7Cb57831c773324018bb9508d8b8735fd4%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637462153217869399%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=Lnv9RSzYGcVy%2BxOn58qa5zd6NMqzQT1sfks2PMrgFNI%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe
I am not familiar with this unit but you have 3 different things to isolate. You may have bubbles in the detector. refreshing the cell or line with water than lots of methanol then water (all degassed/sparged) would treat that. It doesn't sound like an actual peak shape problem from fluid connections but a bad connection can help put in bubbles etc. Finally you can check electrical connections including the outlet connections if you use those. This sounds like bubbles or noise. Hopefully someone with knowledge of this actual unit will respond later in the day. All the best, Eric D. Eric Anderson Mass Spectrometry Facility, NIDDK NIH Building 8 Room B2A19, 9000 Rockville Pike, Bethesda MD, 20892 USA (301)-496-7546
________________________________________ From: OKARVI, SUBHANI MOHAMMAD <email obscured>> Sent: 14 January 2021 02:28 To: <email obscured> Subject: Re: [ABRF Discussion Forum] Shimadzu HPLC issue Hi there, I am having some peak shape issues, with the Shimadzu HPLC system SPD-10A VP UV detector. S.E. and R.E. values (mv) are keep changing and blinking on the system monitor. The D2 lamp used time is 1192 hours. Any suggestion regarding this issue will be appreciated. Subi โโ View topic http://list.abrf.org/r/topic/2Qo0Gfkx0SmFvjyOEjTy79 Leave group <email obscured>?Subject=Unsubscribe
Hi there, I am having some peak shape issues, with the Shimadzu HPLC system SPD-10A VP UV detector. S.E. and R.E. values (mv) are keep changing and blinking on the system monitor. The D2 lamp used time is 1192 hours. Any suggestion regarding this issue will be appreciated. Subi
thanks Kym Francis Faull, Ph.D. Director, Pasarow Mass Spectrometry Laboratory Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA Professor Emeritus on Recall, Department of Psychiatry and Biobehavioral Sciences David Geffen School of Medicine at UCLA Telephone: (310) 206 7881 (office) Telephone: (310) 206 7886 (laboratory) Facsimile: (310) 206 2161 Electronic mail: <email obscured> <email obscured>>
> On Jan 13, 2021, at 2:14 PM, Alex Lobodovsky <email obscured>> wrote: > > Kym: > > I just pinged a Nor Cal MS service guy I know, who likely has experience with this instrument. Most I know are SCIEX people. I'll pass on what I find out. I just rebuild turbo pumps. > > Best, > > Alex Lobodovsky > President > Sertec, Inc. > 1717 Solano Way, suite 18 > Concord, CA 94520 > Tel: 510 430 0833 > www.sertecturboinc.com <http://www.sertecturboinc.com/> > > > On Wed, Jan 13, 2021 at 1:53 PM Kym Faull <email obscured> <email obscured>>> wrote: > Colleagues; A friend in the Bay area needs help from a mass spec repair guy/company/service to help with his old Waters Micromass LCT Premier . The message from Austin is copied below. If you know of an individual or company that has expertise to work on such an instrument please contact Austin Quach (ccโd above). > > Kym > > > Kym Francis Faull, Ph.D. > Director, Pasarow Mass Spectrometry Laboratory > Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA > Professor Emeritus on Recall, Department of Psychiatry and Biobehavioral Sciences > David Geffen School of Medicine at UCLA > Telephone: (310) 206 7881 (office) > Telephone: (310) 206 7886 (laboratory) > Facsimile: (310) 206 2161 > Electronic mail: <email obscured> <email obscured>> > > > > > > > > >> Begin forwarded message: >> >> From: Austin Quach <email obscured> <email obscured>>> >> Subject: help request old instrument repair >> Date: January 13, 2021 at 1:46:30 PM PST >> To: "Faull, Kym F." <email obscured> <email obscured>>> >> >> Hi Dr Faull, >> Here's the message: >> "We just bought an old Waters Micromass LCT Premier from some place in Canada (Spectralabs) and it took them 6 weeks to get it to us and then we spent 7 weeks trying to get it set up and working (via Zoom!) and now they say we need to send it back so they can fix it--they refuse to send anyone here because they think all Americans have Covid. There's no way my boss is going to send it back to them. We are DESPERATE, so if you know of any place or can ask someone else who has had to try to maintain a mass spec on a budget in the Bay Area, that would be incredibly helpful!" >> >> Any help would be appreciated! >> Austin >> >
Colleagues; A friend in the Bay area needs help from a mass spec repair guy/company/service to help with his old Waters Micromass LCT Premier . The message from Austin is copied below. If you know of an individual or company that has expertise to work on such an instrument please contact Austin Quach (ccโd above). Kym Kym Francis Faull, Ph.D. Director, Pasarow Mass Spectrometry Laboratory Jane and Terry Semel Institute for Neuroscience and Human Behavior at UCLA Professor Emeritus on Recall, Department of Psychiatry and Biobehavioral Sciences David Geffen School of Medicine at UCLA Telephone: (310) 206 7881 (office) Telephone: (310) 206 7886 (laboratory) Facsimile: (310) 206 2161 Electronic mail: <email obscured> <email obscured>>
> Begin forwarded message: > > From: Austin Quach <email obscured>> > Subject: help request old instrument repair > Date: January 13, 2021 at 1:46:30 PM PST > To: "Faull, Kym F." <email obscured>> > > Hi Dr Faull, > Here's the message: > "We just bought an old Waters Micromass LCT Premier from some place in Canada (Spectralabs) and it took them 6 weeks to get it to us and then we spent 7 weeks trying to get it set up and working (via Zoom!) and now they say we need to send it back so they can fix it--they refuse to send anyone here because they think all Americans have Covid. There's no way my boss is going to send it back to them. We are DESPERATE, so if you know of any place or can ask someone else who has had to try to maintain a mass spec on a budget in the Bay Area, that would be incredibly helpful!" > > Any help would be appreciated! > Austin >
Hello all, Our group is experiencing a delay in receiving BigDye XTerminator(tm) due to ThermoFisher shipping issues related to this product. We will use all we have on hand before our new stock arrives. Would anyone be able and willing to provide an extra kit (4376484 or 4376487) that they have on hand. I can pay for the reagent and the shipping. I can do a replacement once our reagent arrives as well. Thanks for any help you can offer. Best regards,
Nathan ------------------------------------------------------------------------- Nathan J. Bivens, MS Director, DNA Core Facility University of Missouri 216 Bond Life Sciences Center Columbia, MO 65211 Phone (573) 882-0428 Web DNACore.missouri.edu<dnacore.missouri.edu> Follow the DNA Core on Twitter @MUDNACore<https://twitter.com/MUDNACore>
Hello Hua, I have not -yet- directly compared them. I have used them both for different samples with good success. Functionally, they are equivalent in that they are both negatively charged and have reversed phase characteristics. One obvious difference is convenience. The SDB-RPS material you typically construct yourself for ultimate economy in materials, though I believe there are premade commercial devices available. I have found the self-made tips have a slow flow rate when packed into 200ul pipet tips. So, using a syringe to push liquid through the packed tips is difficult and when spinning in a centrifuge, they take higher G-forces to get the liquid through in a reasonable time. On the other hand, the Waters Oasis MCX are only available in column and plate formats with different amounts of packing material to choose from. The flow rate through the column style MCX is very fast. Spinning in the centrifuge is quick at lower G-force, and if you have only one or a few samples, using a syringe to manually push the liquid through the MCX column is very easy and fast. Your question about recovery will need to be tested with low amounts of sample, which I have not done yet. In the tests I have done using picomoles of a BSA digest, recovery was in the 70-80% range, which I think is typical of SPE workflows. When you pack your own tips using the SDB-RPS you only expose your sample to the plastics and the packing material. With the Oasis products, the column format which I have used, you have an upper and lower frit which holds the resin in place and disperses the sample across the bed as it flows. If there is a difference in recovery between the two, it may show up when desalting low femtomole amounts of peptide digests. The SDB-RPS material is somewhat more amenable to high sensitivity workflows - as demonstrated in numerous publications with e.g. single/few/sorted cells as starting material - because you can construct your own devices that are appropriate for the specialized workflow. The Carr lab has a paper showing some crafty use of tips packed with SDB-RPS etc. tips. However, for routine use where the sample is not limiting the Oasis MCX would be my choice because of convenience, and if you need it high throughput, they have 96-well formats. Best regards, Brian
________________________________ From: ABRF Discussion Forum <email obscured>> on behalf of Ding, Hua <email obscured>> Sent: Wednesday, December 16, 2020 12:16 PM To: <email obscured> <email obscured>> Subject: Re: [ABRF Discussion Forum] What are people's favorite phosphopeptide enrichment methods and protocols? Dear Brian, Thank you so much for your insights! In your own experience, SDB-RPS or Oasis MCX , which one gives better result for peptides desalting? Thanks! Best, Hua -----Original Message----- From: ABRF Discussion Forum <email obscured>> On Behalf Of Hampton, Brian Sent: Friday, December 11, 2020 4:58 PM To: <email obscured> Subject: [External] Re: [ABRF Discussion Forum] What are people's favorite phosphopeptide enrichment methods and protocols? Hi Noah, I've looked at nanomole digests of BSA using LC/UV so I don't have to worry about SPE prior to MS detection. I was using a 2.1mmID column and a PDA detector. I can direct inject the digest (like in the old days when Edman was the go-to sequencing method) and use that as the expected result e.g. what a chromatogram looks like for 100% recovery of the peptides. Using either formic acid or TFA in the SDB-RPS workflow, less than 1% is in the flow through of the SPE. And no peptides are in the 2% MeCN wash prior to elution. The run of the SPE flow through looks like a blank compared to the direct injection of the digest. Recovery in the NH4OH/MeCN elution is 70-80%. This SPE is using the Oasis MCX not Empore SDB-RPS, 2 x 500ul pulses to elute, but they (Empore and Oasis) should be equivalent given similar bed volumes. A more exhaustive test would be a complex digest. But BSA is commonly used for such testing purposes. So there doesn't seem to be any struggle retaining peptides. The SDB-RPS or MCX retention is via mixed modes. Possibly two modes: Charge based and HILIC based (during loading and initial high organic washing, then charge and reversed phase after that when washing at low organic concentration). Given the high organic concentration, I don't think reversed phase characteristics of the support plays any role in retention during loading. The absence of neutral detergents in the eluates is evidence of that. I'm not certain about the role of charge versus HILIC plays in retention given TFA and FA retain equally well. Andy Alpert might lean toward saying HILIC is less effective in the presence of TFA but that TFA would interfere with interaction between -NH3 groups and the -SO4 on the support, therefore, HILIC is the main retention mechanism during loading. Regardless, peptide capture is excellent and exclusion of uncharged or negatively charged hydrophilic (DNA etc.) and hydrophobic (detergents lipids etc.) is also good. I can't bring myself to use just reversed phase SPE any longer. This mixed mode is so much better because you don't have to worry about detergents and can be tweaked for other purposes so that opens a whole world of upstream sample prep possibilities. For example, I can digest a sample in RIPA buffer, and this method cleans it up. You can layer on to this a differential elution scheme as published here: Kulak et al., โMinimal, Encapsulated Proteomic-Sample Processing Applied to Copy-Number Estimation in Eukaryotic Cells.โ DOI: 10.1038/nmeth.2834. They describe a 3-step fractionation of the SDB-RPS retained peptides using a combination of salt and organic in increasing amounts with the final elution with NH4OH/MeCN. You must get the extensive supplementary info for this article for the complete methods and data. To remove the +1 charged junk you mention, would be a matter of finding the right organic solvent composition and ionic strength to wash before elution. I've not tried doing this, but if you have reproducible +1 junk to follow then look at the Kulak paper. Their first eluate for the three-step method is 100mM Ammonium Formate, 40% MeCN, 0.5% Formic Acid. A goodly number of peptides come off in this. So, tweaking downward from those conditions might wash away the +1 junk. Best Regards, Brian ________________________________ From: ABRF Discussion Forum <email obscured>> on behalf of Noah D4 <email obscured>> Sent: Friday, December 11, 2020 3:34 PM To: <email obscured> <email obscured>> Subject: Re: [ABRF Discussion Forum] What are people's favorite phosphopeptide enrichment methods and protocols? CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email system. Whether the sender is known or not known, hover over any links before clicking and use caution opening attachments. Brian, Thanks for your input. I never saw that second easyphos paper or ignored it if I did. Are there any classes of peptides that SDB-RPS struggles to recover? Can it replace C18 for all or most peptide sample clean-up? We see a lot of 1+ junk in IP's and some phosphopeptide enriched samples. I'd love to make it disappear. Noah โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F46wNdpyhY5fEY81J33plFZ&data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2aaf26d9543c45aa863d08d8a1e65d56%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637437358077718325%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=k2rwWC7nUXGPe5jHQQtvqgDQbJRaYu%2F%2BvDpsFygbSks%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F70CXWCMcwcZyAArCvaZDPB&data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2aaf26d9543c45aa863d08d8a1e65d56%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637437358077718325%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=CLtqj7KJa2qZnCco36fgWlds9pkRbhDgUnrYwDirFjs%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe ** This email originated from an EXTERNAL sender to CHOP. Proceed with caution when replying, opening attachments, or clicking links. Do not disclose your CHOP credentials, employee information, or protected health information to a potential hacker**. โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F2XrKjGGH1DIM70Je74VqJq&data=04%7C01%7Cbhampton%40som.umaryland.edu%7C2aaf26d9543c45aa863d08d8a1e65d56%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637437358077718325%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=ftgA%2FJG766WavDUPKNnZ8WKWqxeSOl9EOI5IYzANtz8%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe
Dear Brian, Thank you so much for your insights! In your own experience, SDB-RPS or Oasis MCX , which one gives better result for peptides desalting?
Thanks! Best, Hua -----Original Message----- From: ABRF Discussion Forum <email obscured>> On Behalf Of Hampton, Brian Sent: Friday, December 11, 2020 4:58 PM To: <email obscured> Subject: [External] Re: [ABRF Discussion Forum] What are people's favorite phosphopeptide enrichment methods and protocols? Hi Noah, I've looked at nanomole digests of BSA using LC/UV so I don't have to worry about SPE prior to MS detection. I was using a 2.1mmID column and a PDA detector. I can direct inject the digest (like in the old days when Edman was the go-to sequencing method) and use that as the expected result e.g. what a chromatogram looks like for 100% recovery of the peptides. Using either formic acid or TFA in the SDB-RPS workflow, less than 1% is in the flow through of the SPE. And no peptides are in the 2% MeCN wash prior to elution. The run of the SPE flow through looks like a blank compared to the direct injection of the digest. Recovery in the NH4OH/MeCN elution is 70-80%. This SPE is using the Oasis MCX not Empore SDB-RPS, 2 x 500ul pulses to elute, but they (Empore and Oasis) should be equivalent given similar bed volumes. A more exhaustive test would be a complex digest. But BSA is commonly used for such testing purposes. So there doesn't seem to be any struggle retaining peptides. The SDB-RPS or MCX retention is via mixed modes. Possibly two modes: Charge based and HILIC based (during loading and initial high organic washing, then charge and reversed phase after that when washing at low organic concentration). Given the high organic concentration, I don't think reversed phase characteristics of the support plays any role in retention during loading. The absence of neutral detergents in the eluates is evidence of that. I'm not certain about the role of charge versus HILIC plays in retention given TFA and FA retain equally well. Andy Alpert might lean toward saying HILIC is less effective in the presence of TFA but that TFA would interfere with interaction between -NH3 groups and the -SO4 on the support, therefore, HILIC is the main retention mechanism during loading. Regardless, peptide capture is excellent and exclusion of uncharged or negatively charged hydrophilic (DNA etc.) and hydrophobic (detergents lipids etc.) is also good. I can't bring myself to use just reversed phase SPE any longer. This mixed mode is so much better because you don't have to worry about detergents and can be tweaked for other purposes so that opens a whole world of upstream sample prep possibilities. For example, I can digest a sample in RIPA buffer, and this method cleans it up. You can layer on to this a differential elution scheme as published here: Kulak et al., โMinimal, Encapsulated Proteomic-Sample Processing Applied to Copy-Number Estimation in Eukaryotic Cells.โ DOI: 10.1038/nmeth.2834. They describe a 3-step fractionation of the SDB-RPS retained peptides using a combination of salt and organic in increasing amounts with the final elution with NH4OH/MeCN. You must get the extensive supplementary info for this article for the complete methods and data. To remove the +1 charged junk you mention, would be a matter of finding the right organic solvent composition and ionic strength to wash before elution. I've not tried doing this, but if you have reproducible +1 junk to follow then look at the Kulak paper. Their first eluate for the three-step method is 100mM Ammonium Formate, 40% MeCN, 0.5% Formic Acid. A goodly number of peptides come off in this. So, tweaking downward from those conditions might wash away the +1 junk. Best Regards, Brian ________________________________ From: ABRF Discussion Forum <email obscured>> on behalf of Noah D4 <email obscured>> Sent: Friday, December 11, 2020 3:34 PM To: <email obscured> <email obscured>> Subject: Re: [ABRF Discussion Forum] What are people's favorite phosphopeptide enrichment methods and protocols? CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email system. Whether the sender is known or not known, hover over any links before clicking and use caution opening attachments. Brian, Thanks for your input. I never saw that second easyphos paper or ignored it if I did. Are there any classes of peptides that SDB-RPS struggles to recover? Can it replace C18 for all or most peptide sample clean-up? We see a lot of 1+ junk in IP's and some phosphopeptide enriched samples. I'd love to make it disappear. Noah โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F46wNdpyhY5fEY81J33plFZ&data=04%7C01%7Cbhampton%40som.umaryland.edu%7C6b1bdeba277e4a3337bd08d89e14227b%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637433156599931485%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=oTXUQtLC1tEthVFdEBwng470In%2FuYrwsoGPDCZK%2BfyE%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe โโ View topic http://list.abrf.org/r/topic/70CXWCMcwcZyAArCvaZDPB Leave group <email obscured>?Subject=Unsubscribe ** This email originated from an EXTERNAL sender to CHOP. Proceed with caution when replying, opening attachments, or clicking links. Do not disclose your CHOP credentials, employee information, or protected health information to a potential hacker**.
There should not be any non-volatile salts left. There may be some small amount of ammonium formate or trifluoroacetate formed during elution depending on which acid you used in the final wash prior to elution.
Brian ________________________________ From: ABRF Discussion Forum <email obscured>> on behalf of Noah D4 <email obscured>> Sent: Monday, December 14, 2020 10:33 PM To: <email obscured> <email obscured>> Subject: Re: [ABRF Discussion Forum] What are people's favorite phosphopeptide enrichment methods and protocols? CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email system. Whether the sender is known or not known, hover over any links before clicking and use caution opening attachments. Brian, Thanks again for all the info and discussion. After drying the NH4OH/MeCN eluate, is it sufficiently salt-free for mass spec analysis (I don't use a trap column)? Noah โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F3q3edFho0HTGIFVj6ln6lL&data=04%7C01%7Cbhampton%40som.umaryland.edu%7C257f5a8e0df244ecf9e508d8a0aa316b%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637436000248526326%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=nREnGGCBTg9J5eil8JDG3SLdhTtKOCLvr3VNZJSWBE4%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe
Brian, Thanks again for all the info and discussion. After drying the NH4OH/MeCN eluate, is it sufficiently salt-free for mass spec analysis (I don't use a trap column)?
Noah
Hi Noah, I've looked at nanomole digests of BSA using LC/UV so I don't have to worry about SPE prior to MS detection. I was using a 2.1mmID column and a PDA detector. I can direct inject the digest (like in the old days when Edman was the go-to sequencing method) and use that as the expected result e.g. what a chromatogram looks like for 100% recovery of the peptides. Using either formic acid or TFA in the SDB-RPS workflow, less than 1% is in the flow through of the SPE. And no peptides are in the 2% MeCN wash prior to elution. The run of the SPE flow through looks like a blank compared to the direct injection of the digest. Recovery in the NH4OH/MeCN elution is 70-80%. This SPE is using the Oasis MCX not Empore SDB-RPS, 2 x 500ul pulses to elute, but they (Empore and Oasis) should be equivalent given similar bed volumes. A more exhaustive test would be a complex digest. But BSA is commonly used for such testing purposes. So there doesn't seem to be any struggle retaining peptides. The SDB-RPS or MCX retention is via mixed modes. Possibly two modes: Charge based and HILIC based (during loading and initial high organic washing, then charge and reversed phase after that when washing at low organic concentration). Given the high organic concentration, I don't think reversed phase characteristics of the support plays any role in retention during loading. The absence of neutral detergents in the eluates is evidence of that. I'm not certain about the role of charge versus HILIC plays in retention given TFA and FA retain equally well. Andy Alpert might lean toward saying HILIC is less effective in the presence of TFA but that TFA would interfere with interaction between -NH3 groups and the -SO4 on the support, therefore, HILIC is the main retention mechanism during loading. Regardless, peptide capture is excellent and exclusion of uncharged or negatively charged hydrophilic (DNA etc.) and hydrophobic (detergents lipids etc.) is also good. I can't bring myself to use just reversed phase SPE any longer. This mixed mode is so much better because you don't have to worry about detergents and can be tweaked for other purposes so that opens a whole world of upstream sample prep possibilities. For example, I can digest a sample in RIPA buffer, and this method cleans it up. You can layer on to this a differential elution scheme as published here: Kulak et al., โMinimal, Encapsulated Proteomic-Sample Processing Applied to Copy-Number Estimation in Eukaryotic Cells.โ DOI: 10.1038/nmeth.2834. They describe a 3-step fractionation of the SDB-RPS retained peptides using a combination of salt and organic in increasing amounts with the final elution with NH4OH/MeCN. You must get the extensive supplementary info for this article for the complete methods and data. To remove the +1 charged junk you mention, would be a matter of finding the right organic solvent composition and ionic strength to wash before elution. I've not tried doing this, but if you have reproducible +1 junk to follow then look at the Kulak paper. Their first eluate for the three-step method is 100mM Ammonium Formate, 40% MeCN, 0.5% Formic Acid. A goodly number of peptides come off in this. So, tweaking downward from those conditions might wash away the +1 junk. Best Regards, Brian
________________________________ From: ABRF Discussion Forum <email obscured>> on behalf of Noah D4 <email obscured>> Sent: Friday, December 11, 2020 3:34 PM To: <email obscured> <email obscured>> Subject: Re: [ABRF Discussion Forum] What are people's favorite phosphopeptide enrichment methods and protocols? CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email system. Whether the sender is known or not known, hover over any links before clicking and use caution opening attachments. Brian, Thanks for your input. I never saw that second easyphos paper or ignored it if I did. Are there any classes of peptides that SDB-RPS struggles to recover? Can it replace C18 for all or most peptide sample clean-up? We see a lot of 1+ junk in IP's and some phosphopeptide enriched samples. I'd love to make it disappear. Noah โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F46wNdpyhY5fEY81J33plFZ&data=04%7C01%7Cbhampton%40som.umaryland.edu%7C6b1bdeba277e4a3337bd08d89e14227b%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637433156599931485%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=oTXUQtLC1tEthVFdEBwng470In%2FuYrwsoGPDCZK%2BfyE%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe
Brian, Thanks for your input. I never saw that second easyphos paper or ignored it if I did. Are there any classes of peptides that SDB-RPS struggles to recover? Can it replace C18 for all or most peptide sample clean-up? We see a lot of 1+ junk in IP's and some phosphopeptide enriched samples. I'd love to make it disappear.
Noah
Hi Noah, Since you are having good results w/ HeLa, but not the fat tissue I would suspect sample prep could use some tweaking to accommodate the fat tissue ... though I have no idea what your method is. You could compare your procedure with the EasyPhos method here: Humphrey et al., โHigh-Throughput and High-Sensitivity Phosphoproteomics with the EasyPhos Platform.โ (https://doi.org/10.1038/s41596-018-0014-9). It may be that using SDC, and SDB-RPS SPE clean up method could improve your results. I've used the EasyPhos protocol a couple of times on cultured cells with good success, but not on tissue. However, I use SDB-RPS extensively for SPE because it eliminates neutral and negatively charged/hydrophobic materials e.g. detergents and phospholipids very well. If you don't like making stage tips, you can swap out the stage tips for Waters Oasis MCX products into the workflow. Best regards, Brian
________________________________ From: ABRF Discussion Forum <email obscured>> on behalf of Noah D4 <email obscured>> Sent: Thursday, December 10, 2020 1:13 PM To: <email obscured> <email obscured>> Subject: [ABRF Discussion Forum] What are people's favorite phosphopeptide enrichment methods and protocols? CAUTION: This message originated from a non UMB, UMSOM, FPI, or UMMS email system. Whether the sender is known or not known, hover over any links before clicking and use caution opening attachments. Hi, Despite having worked in this area for a long time, I'm struggling with a few finer points of phosphopeptide enrichment. 1. I thought our TiO2 and newer CaTiO3 methods were pretty fool-proof, but I have some new people struggling to get high reproducibility. Do people have experience with some of the pre-packaged, optimized systems, like the Pierce High Select Fe-NTA columns? How about magnetic bead systems vs spin-filters vs batch? Does anyone have a clear preference either for ease of use or robustness and efficiency? 2. We have been working with some samples derived from fat tissue and are getting horrible phosphopeptide enrichment. A positive control with equal amount of HeLa peptides looks fine. Anyone have any thoughts why this should be? I would have thought that any lipids, including phospholipids would be lost to the C18 during pre-enrichment clean up. Thanks, Noah โโ View topic https://nam11.safelinks.protection.outlook.com/?url=http%3A%2F%2Flist.abrf.org%2Fr%2Ftopic%2F4FtJ9Uj9YPGbyPqQsRnV8W&data=04%7C01%7Cbhampton%40som.umaryland.edu%7C914c2b32d4214d20718a08d89d374862%7C717009a620de461a88940312a395cac9%7C0%7C0%7C637432208046099153%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=svpz9J5%2F4aqs1UpT50Fbs5K1rPsYd99GU0OcwaDmP1Q%3D&reserved=0 Leave group <email obscured>?Subject=Unsubscribe
Hi, Despite having worked in this area for a long time, I'm struggling with a few finer points of phosphopeptide enrichment. 1. I thought our TiO2 and newer CaTiO3 methods were pretty fool-proof, but I have some new people struggling to get high reproducibility. Do people have experience with some of the pre-packaged, optimized systems, like the Pierce High Select Fe-NTA columns? How about magnetic bead systems vs spin-filters vs batch? Does anyone have a clear preference either for ease of use or robustness and efficiency? 2. We have been working with some samples derived from fat tissue and are getting horrible phosphopeptide enrichment. A positive control with equal amount of HeLa peptides looks fine. Anyone have any thoughts why this should be? I would have thought that any lipids, including phospholipids would be lost to the C18 during pre-enrichment clean up.
Thanks, Noah
Hiya Frances ...just checking back in again (hadnt heard from you last email).
Cordially, cvic On Fri, Nov 27, 2020 at 12:45 PM Cvic Innocent <email obscured>> wrote: > Hey Frances > > Happy (late) Thanksgiving and poking my head in to say I hope youre well :] > > Cordially, > cvic > > On Wed, Aug 12, 2020 at 9:35 AM Frances Weis-Garcia < > <email obscured>> wrote: > >> If anyone had to lay someone off in the NYC area, one of our cores could >> a TEMP through the end of the year. I know it is not ideal, but in this >> environment it will give someone a foot in the door at MSK, a job for a few >> months and a good reference should they work well in the team. >> >> If you know of someone, please reach out to me at <email obscured> / >> 646-888-2354. >> >> Thanks, >> >> Frances >> โโ >> View topic http://list.abrf.org/r/topic/6vFRIg4ZDAcI0IT6jyFHOV >> Leave group <email obscured>?Subject=Unsubscribe >> >>
Building a Better Core: Best Practices for Shared Resources - the MAD SSCi 2021 Annual Meeting in 12 Parts - join monthly (2nd Tuesday at 12:00 pm ET) for a series of sessions on timely topics. See the 2021 agenda here: https://madssci.abrf.org/program/ Register: https://us02web.zoom.us/meeting/register/tZYtde2hqzsoE9yk4Cpj0bVZfmXq8ebcuKJG
Dear ABRF members, I would welcome any suggestions on easy options for creating an in-house/potentially open-access database of peptide spectra (acknowledging that the PeptideAtlas has been around for ages, but in house data seems more pertinent). In brief, to save time looking for suitable peptides for targeted proteomics, I am looking for a suitable platform to search in-house data sets (as the most relevant source). Currently, the use of Skyline with a selection of libraries form earlier measurements (+ Prosite) gives a reasonable overview, although not necessarily the frequency of peptide detection. Previously I recall having heard presentations from Lennart Martens on cataloguing all their data, and at an ASMS meeting (~2012, how time flies!) there was a presentation on a software โSliceโ (Manor Askenazi), demonstrating that he could query 2000 Orbitrap files on a $400 Radioshack server. As other reference sources, Galaxy and the MaxQuant database have been useful, plus there have been a number publications with useful accompanying shiny apps. Currently my main data analysis platforms are Proteome discoverer/Mascot and MaxQuant. (It is a long time since I last used the TPP) Thank you in advance for any suggestions Stay safe and healthy Best regards Robert Robert Moulder, PhD Turku Bioscience Biocity, Tykistรถkatu 6 A FI-20520 Turku, Finland
Plan to join us tomorrow (12/2) for the: ABRF December Virtual Members' Town Hall 1:00 pm ET Register here: https://us02web.zoom.us/meeting/register/vJIud-Ctpz4pxcSvEcd9o9wERhGoTGtNFg Along with general updates on ABRF activities, tomorrow's program will feature a presentation from the ABRF DNA Sequencing Research Group (DSRG) on: โComparative Evaluation of Hybrid Capture Methods for Targeted RNA Sequencingโ Presented by: DSRG Chair Zach Herbert with the Molecular Biology Core Facilities at the Dana-Farber Cancer Institute will provide an update on the Group's activities and plans for 2021.
Thanks, Kevin -- Kevin L. Knudtson, Ph.D. University of Iowa Director, IIHG Genomics Division 116B EMRB Iowa City, IA 52242 TEL: 319-335-7251 FAX: 319-335-6737 E-mail: <email obscured>