Defeating Major Contaminants in Fe3+- Immobilized Metal Ion Affinity Chromatography (IMAC) Phosphopeptide Enrichment

Abstract

Here we demonstrate that biomolecular contaminants, such as nucleic acid molecules, can seriously interfere with immobilized metal ion affinity chromatography (IMAC)-based phosphopeptide enrichments. We address and largely solve this issue, developing a robust protocol implementing methanol/chloroform protein precipitation and enzymatic digestion using benzonase, which degrades all forms of DNA and RNA, before IMAC-column loading. This simple procedure resulted in a drastic increase of enrichment sensitivity, enabling the identification of around 17,000 unique phosphopeptides and 12,500 unambiguously localized phosphosites in human cell-lines from a single LC-MS/MS run, constituting a 50% increase when compared with the standard protocol. The improved protocol was also applied to bacterial samples, increasing the number of identified bacterial phosphopeptides even more strikingly, by a factor 10, when compared with the standard protocol. For E. coli we detected around 1300 unambiguously localized phosphosites per LC-MS/MS run. The preparation of these ultra-pure phosphopeptide samples only requires marginal extra costs and sample preparation time and should thus be adoptable by every laboratory active in the field of phosphoproteomics.

Fig. 1.

An optimized sample preparation protocol to tackle main contaminants in Fe³⁺-IMAC phosphopeptides enrichment. Human and bacterial samples can be prepared using the same protocol before Fe³⁺-IMAC column phospho-enrichment. Key steps implemented are the digestion of nucleic acid containing biomolecules after cell lysis, followed by methanol/chloroform protein precipitation. These optimizations resulted in substantial depletion of background, concomitant with an improvement of the purity of the phospho-enriched samples, leading to strong increases in the number of detected phosphorylation events.

Fig. 2.

Comparison between the standard and optimized protocol for column-based Fe³⁺-IMAC phosphopeptide enrichment. A, B, When comparing UV-traces corresponding to human cell-line samples prepared by the standard protocol (A) and optimized protocol (B), an important decrease in the UV signal of the on-column retained molecules (annotated R) is observed. Indeed, with the optimized protocol the phosphopeptide UV-signal corresponds to less than 0.3% of the signal of the flow-through fraction (annotated FT). C, The decrease of the UV trace of the retained material corresponds to an increase in phosphopeptide sample purity through removal of the main interferents, namely nucleic acid containing biomolecules. The presented new protocol effectively eliminates these contaminants by enzymatic digestion, as also attested by the decrease of the proportion of MS2 spectra containing the peak of m/z 330.06, a fragment ion marker of nucleic acid containing biomolecules. D, The optimized protocol enables a remarkable increase in the number of identified protein phosphorylations, even if the enrichment specificity was already around 95% using the standard protocol (at the PSM level, D). E, F, For the human samples the number of unique phosphopeptides (E) and unique class I phosphosites (F) increased by 50% when comparing phospho-enriched samples derived from unstimulated HeLa and HEK293 cell-lines, to reach around 17,000 phosphopeptides and 12,500 class I phosphosites identified per LC MS/MS run. G, Finally, using the new protocol the percentage of multiply phosphorylated peptides identified increased substantially, to about 40% for both cell-lines, doubling the numbers observed using the standard protocol.

Fig. 3.

Improved detectability of bacterial phosphopeptides due to enhanced sample preparation. A, B, Improvements by the optimized workflow were even more spectacular when applied to E. coli samples, as visible by the important reduction of the “retained signal” in the UV-trace profile. C, The protein precipitation step alone did not enable the removal of nucleic acid containing biomolecules (C, annotated std + prec.) but resulted in an increase of the number of phosphopeptides identified and proved to be necessary. D, strikingly this reduced UV peak translated into a substantial increase in the total-ion-current chromatograms (TIC) in the LC-MS/MS runs, which we attribute to an increased phoshopeptide sample purity, resulting in better ionization efficiencies, as the huge background of negatively charged nucleic acid containing biomolecules is depleted in the optimized protocol. E, The increase of phosphopeptide sample purity obtained after preparation with the optimized protocol led ultimately to confounding increases in the number of identified phosphorylation events: numbers of E. coli unique phosphopeptides and class I phosphosites identified increased by a factor 10 when compared with the standard protocol.