LC Separation in the Proteomics Workflow: Why You Can’t Afford to Ignore It

I recently saw a tweet polling the proteomics community on the relative importance, price, and technical difficulty of mass spectrometry-based proteomics: sample prep, liquid chromatography (LC), mass spectrometry (MS), and data processing.

Cue a characteristically lively debate where everyone defends their most beloved stage of the pipeline as the most crucial aspect of proteomics… ultimately, we find ourselves with a few takeaways.

First, depending on a variety of factors, the entire proteomics workflow can be challenging, time-consuming, and expensive.

And perhaps more importantly, each stage is best captured by a computer science maxim stolen to describe just about everything from business to — my personal favorite — pizza making:

Garbage In = Garbage Out

You can’t expect to get good answers without a solid experimental design and, on top of this, there is a cascade effect.

If you start your day with bad sample prep (like samples that are incompatible with your separation, or degraded, etc.) you might as well just go home and take a nap.

At the end of the day, you’ll have the same amount of data to show for it, but at least you’re well rested.

While mass spectrometry often represents the lion’s share of startup costs for a new proteomics lab and tends to generate the most buzz (along with data analysis) at scientific conferences, the LC separation can make or break your lab’s ability to generate deep biological insights.

This tutorial by Lenčo et al.. contains a thorough exploration of the various factors affecting separation quality in reversed-phase LC of peptides.¹

In the tutorial, they argue a fundamental understanding of separation processes and peptide properties is necessary to fully utilize the current state-of-the-art MS technology.

Deeper proteome coverage and better quantitation

Figure 1. Simulation demonstrating the effect of peak width on peak height. Here are the main reasons why high-quality peptide separations lead to deeper proteome coverage and better quantitation:

1. Sharper peaks have higher intensities, improving sensitivity and decreasing the time needed to acquire quality spectra. The effect of peak width on peak height is illustrated in the Figure 1 (simulation). Peak areas shown are identical.
2. Higher peak resolution reduces the competition for charge during electrospray ionization and reduces ion co-isolation/spectral complexity.²
3. Symmetric peaks facilitate alignment of MS1 and MS2 spectra, improving confidence in IDs, particularly for the complex spectra generated in data-independent acquisition (DIA).
4. Reproducible gradients provide stable retention times and peaks shapes, improving your confidence in IDs and enabling match between runs.

Optimized chromatography

In addition to these benefits, optimized chromatography opens the door to balancing sample throughput with proteome depth across nano, capillary, and micro-flow LC ranges.^3-4

Currently, the Thermo Scientific™ Vanquish™ Neo UHPLC  is the only LC system capable of delivering the chromatographic performance, method versatility, and overall reliability necessary to reach the full potential of current high-resolution accurate-mass mass spectrometers.

Thermo Scientific ProFlow™ XR active pump flow control provides excellent retention time precision from 100 nL/min – 100 µL/min (settable down to 1 nL/min) while optimized split-loop autosampler fluidics limit sample dispersion while ensuring high injection volume linearity, precision and accuracy.

In addition to hardware, it is equally as important to use the correct column for your separation. Thermo Scientific µPAC™ and PepMap™ Neo LC columns deliver comprehensive sample coverage, high column-to-column reproducibility, and flow rate flexibility.⁵

So that’s my take on why you cannot afford to ignore the LC separation in your proteomics workflow.

Do you agree? Leave a comment and let’s have a friendly scientific debate.

And you can follow me on X/Twitter for more #proteomics insights.

References

1. Lenco, J. et al. Reversed-phase liquid chromatography of peptides for bottom-up proteomics: A tutor…
2. Zheng, R. et al. Deep single-shot proteome profiling with a 1500 bar UHPLC system, long fully porous…
3. Bian, Y. et al. Robust, reproducible and quantitative analysis of thousands of proteomes by micro-fl…
4. Zheng, R. et al. Fast, sensitive, and reproducible nano- and capillary-flow LCMS methods for high-th…
5. Stejskal, K. et al. Depp proteome profiling with reduced carryover using superficially porous microf…