Dear Steven

The use of nanobore columns allows reduced flow rates which increases the time for the MS system to respond to initial data and to trigger the MS/MS data collection only when the signal is high enough to obtain usable spectra for sequencing. It also allows multiple scans from the same peak to confirm the data being collected. You must bear in mind that the apparent "single peaks" seen in the peptide elution profiles are often overlapping peaks of more than one peptide that will give overlapping spectra that need to be assessed to determine the best result. Due to the many problems around nanoLC there is a trend to go to slightly higher flow rates (microflowLC)but this requires different pumping systems and can use larger diameter columns.

In metabolomics the compounds are usually small molecules with less complex MS/MS spectra so there is less requirement for the longer data collection times and in many cases the sample sizes are also larger so the sensitivity is also not such an issue.

Dear Steven

The use of nanobore columns allows reduced flow rates which increases the time for the MS system to respond to initial data and to trigger the MS/MS data collection only when the signal is high enough to obtain usable spectra for sequencing. It also allows multiple scans from the same peak to confirm the data being collected. You must bear in mind that the apparent "single peaks" seen in the peptide elution profiles are often overlapping peaks of more than one peptide that will give overlapping spectra that need to be assessed to determine the best result. Due to the many problems around nanoLC there is a trend to go to slightly higher flow rates (microflowLC)but this requires different pumping systems and can use larger diameter columns.

In metabolomics the compounds are usually small molecules with less complex MS/MS spectra so there is less requirement for the longer data collection times and in many cases the sample sizes are also larger so the sensitivity is also not such an issue.

Thanks for your input!! Duncan, I think the simpler MS/MS spectra aspect is a great point. Would you therefore say that fast flow UPLC is more appropriate for metabolomics rather than proteomics?

Regarding lower flow rates though, I believe nano and conventional LC typically have the same linear velocity, so the peaks are just as broad time-wise. Wouldn't this mean that the MS would have the same response time?

PS, I have found it funny that capLC has often fallen between two chairs ("conventional" and nano). 0.5 mm bore columns are often so great to work with! Duncan, can you recommend any papers on use of capLC in proteomics?

I too strongly agree with the previous comments. I would however add that the metabomolics field has been rapidly shifting toward GC-MS and GCxGC-TOF-MS. Indeed, comprehensive two-dimensional GC-MS provides a superior chromatographic resolution of volatile metabolites compared to LC-MS. Also, electron impact (EI) ionization conditions enable the use of searchable mass spectra libraries for the identification of the metabolites in complex mixtures. The low volatility of peptides in the proteomics field cannot take advantage of these analytical platforms. 

That a quite huge question J

I don’t think is a matter of “culture” but more a matter of convenience. In principle, regarding the accuracy of detection, a flow rate “low as possible” is ever the best choice in metabolomics and proteomics too, not much for the MS response which doesn’t change much between high and low flow rate, but for the ionization efficiency and stability of the spray during ESI process (the most common source in LC/MS analysis). Of course if you want to have MS/MS analysis you have a scanning cycle which is longer than MS only, so to have the same number of scans in MS/MS you need larger peaks, but this can be easily address by adapting the method you are using.

On the other hand, lower flow rate may reduce the separation efficiency cuz you use a value that is far away from the optimal flow rate (Van Deemter). This result obviously in peak broadening (so the signal is less intense) and reduced separation efficiency.

The last point (which is the one that play the major difference in my opinion) is the time you required for one analysis. Of course if you want to see a limited number of peaks (whatever it is) and you don’t have much issues about sensitivity than the best way is the fastest one (which it is often the case in targeted metabolomics)

There are more other points that speak for one or the other technique like lifetime of the consumables, instrumental price, environmental impact, starting material availability etc.

Thanks Sébastien for your input! GCGC is definitely to drool over, and too bad it isn't compatible with bio-macromolecules. There are some groups working with trying to combine LC and EI, would be cool if this could work.

Giuseppe, thanks for your input. Regarding lower flow rates, HILIC might be a good choice as the lowest point in the VD is often half of that with silica based RP columns, what do you think about HILIC?

Are you sure that nanospray suffers from more suppression? I'm just thinking that the larger number of smaller droplets create less of a within-drop competition between co-eluting compounds?

I strongly agree with Duncan who gave you the exact reason. complexity of sample is criteria for selection of one of two chromatograph instruments.

Dear Steven, you are right about the ion suppression (my mistake sorry).

Regarding HILIC, still depend on how the stationary phase is build (i.e. particle size and geometry). Now on the market one of the best choice (in my opinion) is to select core-shell particles (HILIC, C18 or any other available SP), however this is not always possible cuz there are not many stationary phases available with this technology and there are few companies providing columns with ID < 2.1 mm.

I have been working both  with HILIC kinetex stationary phase and C18 kinetex and so far they are my favorite SP. Unfortunately, those are not available for small ID columns, but you can use HALO SP for nano-LC. For me Halo are not as good as kinetex, but still 10 time better than a classic stationary phases.

We've just had some great success with microflow on our system having shifted from nanoflow for proteomics.  We tried HALO 0.3 and 0.5mm columns, but settled on a YMC (300um i.d. capillary column) Triart C18 with 1.9um particle size which gave much better peakshape and Rt reproducibility on our system - great peak shape with very low back pressure for such small particle size (typically 3300 psi, 50:50 ACN:H2O at 30 Celcius).  Perhaps a 2-3 fold loss of sensitivity at most from nano, but we can now load 5-10x more mass on column to overcome this.

Hi Giuseppe, I agree that the core-shells are great! Really appreciate you sharing your experiences with the various brands!

Hi David! Really interesting to hear about your experiences with upscaling to capLC. How much do you load in volume (still on to an one-line SPE first, I presume)?

No, you all missed the point. Proteomics people usually count spectra, they don´t bother with poor signal stability, resulting in noisy peaks, as long as peptides are counted correctly. Metabolomics people use peak area or height, and need signal stability. They also care about retention time consistency, since both retention time and peak area response are considered important parameters in the multivariate analysis, such as PCA etc. All this is easier with upscaled systems. This is my oversimplified view of course.😀

Steven,

We don't bother with trap/elute (sample clean up off line beforehand if necessary), its set up as direct injection (though you could trap if you wanted) - usually injecting 1-10uL volume (normally 1-3), about 1-10ug protein on column, only limited by hitting detector saturation really.  Unlike Cato, we certainly are bothered about signal stability for peak area quantitation in proteomics - I don't really see the difference between proteomics and metabolomics analytically.

Hi David! Do you do a desalting prior to injection (e.g zip-tip), or do you let the salt from the sample pass with the void volume through the column and split to it waste?

Would you say that cap LC is the key to a single system for metabolomics/proteomics?