Signal and Charge Enhancement for Protein Analysis by Liquid Chromatography-Mass Spectrometry with Desorption Electrospray Ionization

Abstract

We recently reported the use of desorption electrospray ionization (DESI) as a novel interface to couple high-performance liquid chromatography (HPLC) with mass spectrometry (MS) (Chem. Commun. 2011, 47, 4171). One of the benefits of such an interface is that post-column derivatization of separated analytes can be integrated with ionization via a "reactive" DESI approach in which a derivatizing reagent is doped into the spray solvent. The reactive DESI interface allows analyte desorption/ionization from the end of the chromatographic column with prompt MS detection; a short time delay of ~20 ms was demonstrated. In this study, we extended this application by "supercharging" proteins following HPLC separation using a DESI spray solvent containing supercharging reagents, m-nitrobenzyl alcohol (m-NBA) or sulfolane. Proteins (insulin, ubiquitin, lysozyme and α-lactalbumin) eluted out of the LC column can be supercharged with the protein charge state distributions (CSDs) significantly increased (to higher charge), which would be advantageous for subsequent top-down MS analysis of proteins. Interestingly, supercharging combined with reactive DESI enhances tolerance towards trifluoroacetic acid (TFA), which is known to be a superior additive in the mobile phase for premium peptide/protein chromatographic separation but has severe signal suppression effects for conventional electrospray ionization (ESI). In comparison to electrosonic spray ionization (ESSI), a variant form of ESI, the sensitivity of protein analysis using LC/DESI-MS with the mobile phase containing TFA can be improved by up to 70-fold for lysozyme and α-lactalbumin by including m-NBA in the DESI spray solvent. Presumably, by reducing TFA dissociation in the droplet, supercharging agents lower trifluoroacetate anion concentrations and concomitantly reduce ion pairing to analyte cationic sites. The reduced ion pairing therefore decreases the TFA signal suppression effect. The supercharging capability and the reduction of TFA signal suppression suggest that LC/DESI-MS is a valuable method for protein analysis.

Keywords: Liquid chromatography/mass spectrometry; TFA ion suppression; protein supercharging; reactive DESI.

Figure 1

Scheme showing the online coupling of HPLC with the DESI-MS apparatus. A DESI spray probe was used to sample/ionize the analytes of interests in the eluent directly from the fast flow jet.

Figure 2

DESI mass spectra of 10 μM lysozyme in 0.1% TFA using a spray solvent of CH₃OH/H₂O/HOAc (50:50:1 by volume) (a) without m-NBA and (b) with 50 mM m-NBA; DESI mass spectra of 10 μM ubiquitin in 0.1% TFA using a spray solvent of CH₃OH/H₂O/HOAc (50:50:1 by volume) (c) without m-NBA and (d) with 50 mM m-NBA. The peaks of m/z 700~1100 in (a) were from background leftover.

Figure 3

UV chromatogram showing the HPLC separation of a protein mixture containing insulin, ubiquitin, lysozyme and α-lactalbumin.

Figure 4

Mass spectra of lysozyme following the HPLC separation obtained by (a) ESSI analysis after splitting the eluent (split ratio: 100:1), (b) DESI analysis of the jet eluent using the spray solvent of CH₃OH/H₂O/HOAc (50:50:1 by volume), (c) DESI analysis of the jet eluent using the spray solvent of CH₃OH/H₂O/HOAc (50:50:1 by volume) containing 50 mM m-NBA, and (d) DESI analysis of the jet eluent using the spray solvent of CH₃OH/H₂O/HOAc (50:50:1 by volume) containing 200 mM sulfolane. The asterisk labeled peaks in (c) probably correspond to the protein adduct ions with m-NBA.

Figure 5

Mass spectra of α-lactalbumin following the HPLC separation obtained by (a) ESSI analysis after splitting the eluent (split ratio: 100:1), (b) DESI analysis of the jet eluent using the spray solvent of CH₃OH/H₂O/HOAc (50:50:1 by volume), (c) DESI analysis of the jet eluent using the spray solvent of CH₃OH/H₂O/HOAc (50:50:1 by volume) containing 50 mM m-NBA, and (d) DESI analysis of the jet eluent using the spray solvent of CH₃OH/H₂O/HOAc (50:50:1 by volume) containing 200 mM sulfolane. The asterisk labeled peaks in (c) probably correspond to the protein adduct ions with m-NBA.