Assessment of capillary electrophoresis TOF MS for a confident identification of peptides

Abstract

CE on-line coupled to orthogonal accelerated TOF-MS (CE-oa-TOF-MS) is an emerging technique offering efficient charge-to-mass-based separations, as well as accurate and high-resolution mass measurements. Here, we investigated the main factors influencing the analysis of low molecular mass peptides using a sheath-flow electrospray ionization interface and several neuropeptides as model compounds. Moderate fragmentor voltage values of the oa-TOF-MS were crucial to maximize the production of molecular ions for optimum sensitivity precluding molecular fragmentation. However, the major fragments provided specific information that may result valuable for confirmatory purposes. Advantages and disadvantages of adding internal mass references to the sheath liquid for continuous mass-to-charge recalibration during the electrophoretic separations were discussed with regard to mass accuracy and sensitivity. Furthermore, several instrumental modes related to mass resolution were also examined. Finally, the method was validated for quantitative analysis of the studied neuropeptides in terms of repeatability, linearity and LODs. The results obtained using CE-oa-TOF-MS were compared with those obtained using CE coupled to other mass spectrometers. In addition to the simplicity and reliability on the identification, CE-oa-TOF-MS allowed improved repeatability and an around 10-fold improvement in sensitivity with respect to conventional reflectron TOF and ion trap mass analyzers.

Transient isotachophoresis in on‐line solid phase extraction capillary electrophoresis time‐of‐flight‐mass spectrometry for peptide analysis in human plasma

“…Internal mass recalibration was satisfactory selecting the single molecular ions of phthalic anhydride and diisooctyl phthalate (i.e. 149.0233 and 391.2843, respectively), which were commonly found in the background mass spectra [26].…”

“…A 6220 oa-TOF LC/MS mass spectrometer (Agilent technologies) with the orthogonal G1603A sheath-flow interface (Agilent Technologies) was coupled to the CE system [26]. The sheath liquid was delivered at a flow rate of 3.3 mL/min by a KD Scientific 100 series infusion pump.…”

“…The oa-TOF mass spectrometer was operated in positive mode using the following parameters: capillary voltage 4000 V, drying gas temperature 2001C, drying gas flow rate 4 L/min, nebulizer gas 7 psig, fragmentor voltage 215 V, skimmer voltage 60 V, OCT 1 RF Vpp voltage 300 V [26]. Unless otherwise indicated, data were collected in profile at 1 spectrum/s (aprox.…”

“…Repeatability values were calculated as a percentage of the RSD (%RSD) of peak area, migration time or experimental monoisotopic molecular mass (M). M accuracy was expressed in ppm as an average of the ratio between the difference of the experimental M and the theoretical M and the theoretical M [26].…”

“…In recent years, orthogonal acceleration reflectron TOF (oa-TOF) mass spectrometers are being regarded as an excellent choice for CE-MS because they provide high resolution and mass accuracy at a reasonable cost, as well as reduced bench space, user friendly operation, high acquisition rates and potentially unlimited mass acquisition ranges in a single run [25,26].…”