Nano-electrospray and microbore liquid chromatography-ion trap mass spectrometry studies of copper complexation with MHC restricted peptides

Abstract

The formation of copper/peptide complex ions by nano-electrospray and microbore HPLC-electrospray mass spectrometry has been investigated for major histocompatibility complex (MHC) class I and class II restricted peptides. Post-column addition of copper(II) acetate following microbore HPLC-MS separation was carried out using a mixing T-piece or via the sheath flow inlet of the electrospray source. Optimal analytical conditions for copper complex ion formation were determined by variation of copper concentration, pH, nebulization gas supply and spray voltage. Tandem mass spectrometry of copper/peptide complex ions provides peptide sequence information and insight into the peptide chelation sites. Copper associated y fragment ions dominate the product ion spectrum for non-histidine containing peptides, but both b and y copper complex ions were observed for the histidine containing MHC class I associated peptide gp70.

Electrospray mass spectrometric study of anionic complexes of enkephalins with Cu(II): regioselective distinction of Leu/Ile at the C‐terminus induced by metal reduction

“…In addition to the above interpretation of the behavior of these Cu II complexes in the ESI source, we explored the possibility of direct Leu/Ile differentiation from the in‐source dissociations of such complexes by liquid chromatography coupled with an ESI ion trap mass spectrometer. In this experiment the complexation metal reagent is introduced after the analytical column by use of a syringe pump 31,43. Similar trends were obtained under LC/ESI‐MS conditions as in the direct infusion mode, even though the composition of the CH 3 CN/H 2 O solvent mixture is modified during the separation gradient.…”

“…The metallation of these peptides was explored in LC/MS experiments by post‐column addition of a solution of a divalent metallic cation salt introduced using a syringe pump via a tee‐piece located between the analytical column and the ESI source. The CuCl 2 salt solution (32 mM, flow rate 1200 μL/min) was adjusted to pH 11 by adding NH 4 OH for an improvement of the metallation yield, as described by Creaser and co‐workers 31. The YGGFL and YGGFI enkephalins (24 pmol/μL of each peptide, 10 μL injected) were separated on a C 18 column (Zorbax stable Bond, 2.1 × 150 mm) by an elution gradient from 80–20% to 50–50% (H 2 O/CH 3 CN) in 8 min.…”

Structural analysis of oligosaccharides by atmospheric pressure matrix‐assisted laser desorption/ionisation quadrupole ion trap mass spectrometry

“…As a consequence, under typical operating conditions, only a single stage of tandem mass spectrometry may be available using these techniques, limiting their scope for the analysis of complex oligosaccharides. In contrast, it has been demonstrated that the quadrupole ion trap is a useful tool for the analysis of biomolecules such as peptides,31–33 oligonucleotides34,, 35 and oligosaccharides,13,, 14 through the use of successive stages of trapping and CAD to reveal structural details that cannot be resolved by MS/MS alone.…”

On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides

“…There was also negligible leaching of Cu(II) ions, indicated by the absence of Cu(II)-peptide adducts, which are detectable by ESI [29]. In addition, Cu(II) ion leakage was minimised by underloading the metal ion on the Poros media.…”

“…This peptide does not contain a histidine residue, but the sequence includes tryptophan. It has previously been shown that the tryptophan indole side chain shows some affinity for Cu(II) ions [29], but to a lesser extent than histidine. In comparison to the mass spectrum obtained without IMAC separation (Fig.…”