Internal Standard Quantification Using Tandem Mass Spectrometry of a Tryptic Peptide in the Presence of an Isobaric Interference

Abstract

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/z mass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.

Structural analysis of a compound despite the presence of an isobaric interference by using in‐source Collision Induced Dissociation and tandem mass spectrometry

“…The MS/MS spectrum of Int‐Pept obtained at 25 V (spectrum a; see top left Figure 1) exhibits a number of fragment ions, whereas Arg‐Pept does not show any sign of fragmentation at this collision voltage (spectrum c; see bottom left Figure 1). This is because, in general, the cleavage of a peptide bond between an arginine residue and the adjacent amino acid requires more energy 13,30,31 . At a higher collision voltage (45 V), all the precursor ions of Int‐Pept are fragmented (spectrum d), whereas the precursor ions of Arg‐Pept (spectrum f) are not fully fragmented yet and give rise mainly to fragment ions with LMs.…”

“…SY curves of pure compounds are indeed usually sigmoidal. However, in the presence of an iso‐baric/meric interference, the SY curve may be distorted, showing a hump or even a plateau if the fragmentation parameters of the two compounds are different enough 6,8,12–14 . In this context, SY curves were used as an indicator of iso‐baric/meric contamination and to provide information not only for quantification purposes but also for structural analysis.…”

“…Thanks to the presence of this plateau, we have shown that the structural analysis of two 6 and, more recently, up to three isobaric compounds 14 can be performed. Two kinds of information can be obtained from this plateau: (1) its vertical position provides the relative gas‐phase abundances of the mixture of compounds, 6,8,12–15 and (2) the corresponding collision voltage provides valuable information to perform a new strategy, which we have called the gas‐phase collisional purification (GPCP) 15 . The latter was shown to allow for the collisional purification of the analyte from the interference and for its structural analysis as though it were free from interference 12–15 .…”

“…Two kinds of information can be obtained from this plateau: (1) its vertical position provides the relative gas‐phase abundances of the mixture of compounds, 6,8,12–15 and (2) the corresponding collision voltage provides valuable information to perform a new strategy, which we have called the gas‐phase collisional purification (GPCP) 15 . The latter was shown to allow for the collisional purification of the analyte from the interference and for its structural analysis as though it were free from interference 12–15 . This GPCP strategy has also been applied for quantitative analysis in order to (a) determine the degree of purity of a cyclic compound potentially contaminated by its linear topo‐isomeric precursor by using the standard addition method 12,15 and (b) to carry out an internal standard quantification of a compound of interest despite the presence of an isobaric interferent 13 …”

“…The latter was shown to allow for the collisional purification of the analyte from the interference and for its structural analysis as though it were free from interference 12–15 . This GPCP strategy has also been applied for quantitative analysis in order to (a) determine the degree of purity of a cyclic compound potentially contaminated by its linear topo‐isomeric precursor by using the standard addition method 12,15 and (b) to carry out an internal standard quantification of a compound of interest despite the presence of an isobaric interferent 13 …”