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

Written by | Published: 02.06.2023

Is your proteomics data lacking sensitivity? Discover how high-quality peptide separations lead to deeper proteome coverage and better quantitation.

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Key takeaways:

  • Efficient reversed-phase LC separations are critical for deep proteome coverage and reliable peptide quantitation in bottom-up (shotgun) proteomics
  • High-quality, reproducible separations require both optimized LC hardware and efficient LC columns

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

Que 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 Mass Spectrometry 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 it, 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

Here are the main reasons why high-quality peptide separations lead to deeper proteome coverage and better quantitation:

Figure 1. Simulation demonstrating the effect of peak width on peak height.
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Figure 1. Simulation demonstrating the effect of peak width on peak height.

  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 Figure 1. Peak areas shown are identical. Historically, MS scan rates were a limiting factor, meaning that narrower peaks were not always better for identifying and quantifying peptides. With the speed of current state of the art systems, such as the Thermo Scientific Orbitrap Astral Zoom mass spectrometer, narrow peaks are almost always preferable.
  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, our Thermo Scientific Vanquish Neo UHPLC System 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. Active flow control and optimized fluidics provide extremely stable separations with high injection linearity, precision, and accuracy even in the low nL/min range.

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

Unlike traditional packed bed columns, micro Pillar Array Columns (µPAC) utilize a unique, microfabricated separation bed that enhances separation performance, boosts columns lifetime6, and yields tighter precision between columns and runs. Figure 2 illustrates how µPAC columns provide narrower peaks than packed bed columns due to the presence of perfectly ordered stationary phase “particles.”

Depiction and corresponding simulation of band broadening in packed bed and µPAC columns. By limiting variability in the physical path each analyte can take across a separation column (A term of the Van Deemter Equation), µPAC yields narrower peaks and superior resolution compared with packed bed columns.

Figure 2. Depiction and corresponding simulation of band broadening in packed bed and µPAC columns. By limiting variability in the physical path each analyte can take across a separation column (A term of the Van Deemter Equation), µPAC yields narrower peaks and superior resolution compared with packed bed columns.

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.

Frequently asked questions

What is active flow control on the Vanquish Neo UHPLC system and why is that important?

Thermo Scientific ProFlow XR active flow control uses a continuous feedback loop between measured flow and piston speed to deliver the programmed flow rate with high precision, rather than relying on piston displacement alone. This is important because it maintains accurate, reproducible gradient delivery and excellent retention time precision across flow rates, regardless of pump seal wear.

How are micro pillar array (µPAC) columns different from traditional packed nano LC columns?

µPAC columns comprise of two silicon wafers – essentially glass microscope slides – fused together with open channels (the column) and pillars (the particles) sandwiched between them. Because the channels and pillars are microfabricated in a process similar to making computer chips, all structures are laid out in a perfectly ordered design. This differs from traditional columns with random packing of spherical particles that leads to heterogeneous channels forming along the column bed. As a result, the analyte bands exiting the µPAC column are narrower and better resolved.

Why is peak width so important to proteome coverage in bottom-up proteomics?

Peak width is one of the key chromatographic metrics that dictates how many peptides co-elute at a given time. In the simplest sense, you can fit a greater number of narrow peaks into a chromatogram without overlapping than you can wide peaks. Fewer overlapping peaks (higher resolution) lead to less electrospray ionization suppression, while narrower peaks yield a higher analyte concentration at any point across the peak, both of which increase sensitivity. Additionally, narrow peaks generally reduce mass spectral complexity, improving coverage and quantitation.

References

  1. Lenčo, J. et al. Reversed-phase liquid chromatography of peptides for bottom-up proteomics: A tutorial. J Proteome Res. 2022, 21, 12, 2846–2892
  2. Zheng, R. et al. Deep single-shot proteome profiling with a 1500 bar UHPLC system, long fully porous columns, and HRAM MS. J. Proteome Res. 2022, 21, 10, 2545-2551
  3. Bian, Y. et al. Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS. Nat Commun, 2020 11, 157
  4. Zheng, R. et al. Fast, sensitive, and reproducible nano- and capillary-flow LCMS methods for high-throughput proteome profiling using the Vanquish Neo UHPLC system hyphenated with the Orbitrap Exploris 480 MS. 2022. Technical Note 000138.
  5. Stejskal, K. et al. Deep proteome profiling with reduced carryover using superficially porous microfabricated nanoLC columns. Anal. Chem. 2022, 94, 46, 15930–15938
  6. Schroeter, C. et al. Evaluating the Robustness of Micro-Pillar Array Columns for Quantitative Proteomics Applications. J. Proteome Res. 2026, 25, 1, 498–505

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Alec Valenta

Written by:

Alec Valenta, Ph.D.

Product Marketing Manager, Chromatography and Mass Spectrometry Division, Thermo Fisher Scientific

Alec Valenta is an HPLC Product Marketing Manager with a Ph.D. in Analytical Chemistry from the University of Michigan where he studied neurochemistry using liquid chromatography mass spectrometry.

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