Sense and Nonsense of Elevated Column Temperature in Proteomic Bottom-up LC–MS Analyses

Abstract

Elevated column temperature represents a simple means for improving chromatographic separation of peptides. Here, we demonstrated the advantages of the column temperature in peptide separation using state-of-the-art columns. More importantly, we also determined how temperature can impair proteomic bottom-up analyses. We found that an elevated temperature in combination with the acidic pH of the mobile phase induced in-column peptide hydrolysis with high specificity to Asp and accelerated five modification reactions of amino acids. The positive effects of temperature dominated in the 30 min long gradients since the column operated at 90 °C provided the largest number of identified peptides and proteins. However, the adverse effects of temperature on peptide integrity in longer liquid chromatography–mass spectrometry (LC–MS) analyses required its reduction to obtain optimum results. The largest number of peptides was identified using the column maintained at 75 °C in 60 min long gradients, at 60 °C in 120 min long gradients, and at 45 °C in 240 min long gradients. Our results indicate that no universal column temperature exists for bottom-up LC–MS analyses. Quite the contrary, the temperature setting must be selected rationally to exploit the full capabilities of the state-of-the-art mass spectrometers in proteomic LC–MS analyses, with the gradient time being a critical factor.

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial

“…The contribution of the longitudinal dispersion becomes apparent at 90 °C at very low flow rates. The plot earlier published by Lenčo et al in Journal of Proteome Research was adapted for the figure.…”

“…Bottom-up LC-MS analyses of samples expected to contain a considerable amount of Pro-rich proteins, such as saliva, 143 should be carried out using a column kept at a temperature of 60 °C and higher to achieve a superior separation of resulting tryptic peptides. 107 Chemical Inertness of Peptides. The chemical reactivity of amino acid residues and stability of peptide bonds during LC-MS analyses is vastly disregarded.…”

“…Upon such conditions, the column is easily transformed into a chemical reactor, resulting in artifacts such as Glu and Gln converted to pyroglutamic acid, dehydration of Asp, and peptide bond cleavage at Asp. 107 An additional source of unwanted in-column reaction is associated with metal traces that induce oxidation, with the most common target being Met residue. 144 Such in-column oxidation can be sorted out by remedies that remove metal traces already discussed in the section above.…”

“…Indeed, peptide separations cannot provide optimum results when carried out close to ambient temperature (Figure 13). 107 For the efficient separation of proteins, the use of high column temperature is absolutely essential (see the section Column Temperature). 193,194 Other Sources of In-Column Band Broadening.…”

“…In the bottom-up proteomics analyses, this manifests itself as narrower peaks of peptides eluted at the initial flow rate (Figure 13). 107 The temperature has also been shown to enhance the recovery of peptides, thus minimizing their carry-over. 393 This applies particularly to hydrophobic peptides.…”