Nano-High-Performance Liquid Chromatography with Online Precleaning Coupled to Inductively Coupled Plasma Mass Spectrometry for the Analysis of Lanthanide-Labeled Peptides in Tryptic Protein Digests

摘要

Low background signals are an indispensable prerequisite for accurate quantification in bioanalytics. This poses a special challenge when using derivatized samples, where excess reagent concentrations are increasing the background signal. Precleaning steps often are time-consuming and usually lead to analyte losses. In this study, a set of labeled model peptides and a protein digest was analyzed using inductively coupled plasma mass spectrometry (ICPMS), coupled to nano ion pairing reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC). In addition, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for peptide identification. Peptides were labeled with lanthanide metals using bifunctional DOTA-based (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) reagents. The resulting metal excess was removed online during nano-HPLC, by trapping the labeled peptides on a C18-precolumn and washing them prior to their elution to the analytical column. Different ion pairing reagents like TFA (trifluoroacetic acid) and HFBA (heptafluorobutyric acid) were used in the study to enhance interactions of the different peptide species with the C18 material of the precolumn. HFBA even allowed the detection of a highly hydrophilic peptide that was not retained using TFA. It was shown that for the mixture of labeled model peptides, even a short 3 min washing step already enhanced the removal of the excess reagents significantly, whereas peptide losses were observable starting with a 10 min washing time. A 6 min washing time was determined to be the best parameter for lowering the lanthanide metal background while maintaining maximum peptide recovery. Alternative precleaning setups using EDTA to enhance the removal of free metal or an offline approach using solid phase extraction did not show promising results. The application of the optimized method to labeled peptides in a lysozyme digest showed results comparable to those obtained with model peptides.