Hello there,
I know that shotgun proteomics relies on high-resolution instruments (i.e. Orbitrap and New QTof systems) with nano-LC flow and nano-ESI mostly, however I only have a reach to a Microflow LC with an older QTof (Waters Micromass Micro). These instruments work fine, however I recently discovered that the resolution of the QTof is slightly higher than 5000 FWHM, which is much lower than today's QTof instruments.
I'm planning on using the SILAC (where isotopic amino acids are used) technique to determine the regulated proteins in my human cell samples. I'm not going to discover a protein which was not discovered before or anything.
How reliable using this LC-QTof system will be in my studies? Will software such as MaxQuant be able to determine the proteins which are regulated in my protein samples, or will the low resolution and high LC flow rate be a problem?
Hello Bora Onat
The quick answer is to give it a try with a SILAC sample you are willing to sacrifice.
SILAC relies on quantifying peptide pairs (one light & one heavy) that are separated by discrete m/z units depending on the isotope used to label the amino acids. If you label with R10 (heavy arginine), then a doubly charged peptide species will be separated by 5 m/z units between the light & heavy. Your "older" QToF should be ok for SILAC work.
It may be the micro flow LC that causes the headache as many peptide pairs will be co-eluting in a given time frame, so the amount of MS1 and MS2 the QToF can generate at high LC flow rates may be the limiting step.
Finally, software (such as MaxQuant) will be able to analyze QToF data - it just depends on the quality of the input data.
So, give it a try & see what happens.
It if works - great! You can then continue with your SILAC MS work.
If it does not work, then you can invest resources into alternatives such as finding a collaboration with a nano-LC orbitrap-MS (or something else!)
Good luck.
Best,
Peter
Hello Peter and thanks for your suggestions. I actually discovered that the LC pump is a microflow pump, but there is a nanoflow splitter cartridge installed to the LC regulator, so we'll probably be able to get nL/min flow.
The issue is with the MS. It is a very old one and the TOF tube is pretty tiny. They say the resolution is lower than a modern TOF's resolution.
Which separation column do you find useful, especially in terms of robustness?
Thanks
Hi,
Offline peptide fractionation can improve the general coverage, TOF resolution & sensitivity should be enhanced by the sample prep and adjusting the chromatographic sweet spot. Additionally desalting and peptide enrichment approaches can also increase the identification sensitivity. Multiplexing using SILAC is very useful for shotgun proteome comparison and that's for sure sensitivity is critical but the chromatographic resolution is important equally. At this point, I would suggest using Biozen PS C18 since 3 um, 250mm length, and the charged surface can improve the resolution. A trap column will contribute much to get stability. high length with small particle size will contribute much to resolution and charge surface repels the cationic tryptic peptides thus gaussian peaks could be obtained.
https://www.phenomenex.com/products/part/00g-4771-aw-21
SPE/Stagetip practices may both purify and enrich your peptides
prior to injection, As I pinpointed if these considerations cannot be applicable or not satisfying combine your workflow with high pH offline fractionation to increase the resolution where TOF's resolution is not enough.
Emir
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