Improved repeatability and matrix‐assisted desorption/ionization – time of flight mass spectrometry compatibility in capillary isoelectric focusing

Abstract

Low repeatability of migration time, peak area, and linearity (pI vs. mobilization time) is a problem often encountered in capillary IEF (cIEF) and is mainly caused by protein precipitation and protein-wall interactions. In order to study the influence of these phenomena, the effect of different classes of additives on repeatability of migration time, peak area and linearity of a mixture of seven model proteins has been investigated. Moreover, the influence of these additives on protein signal suppression in MALDI-TOF MS has been studied. The optimal ampholyte blend (stabilizes pH gradient) to be used depends on the selected UV detection wavelength. All tested ampholyte blends show a significant and comparable signal suppression in MS. The best detergent (to prevent precipitation and wall interaction) should be determined for each sample individually, but generally polyethylene oxide and zwitterionic detergents show good repeatability for migration time (RSD <4.5%) and peak area (majority <10%). The RSD of R(2) is <1.3% for the hydrophilic protein mixture. However, these components cause severe signal suppression in MS. Therefore glucoside detergents should preferably be used for MS coupling. Viscosity-increasing agents (for hydrodynamic wall coating and to minimize diffusion) in particular cellulose derivatives, give good repeatability for migration times (RSD <4.5% at lower concentrations), peak area (except for high concentration methylcellulose and hydroxyethylcellulose all within 7.5%), and correlation (pI vs. migration time), but severe signal suppression is observed in MALDI-TOF MS. Overall, cIEF repeatability and linearity can significantly be improved by adding the appropriate components. However, when the system is coupled to a MALDI-TOF MS, compromises have to be made between high repeatability and linearity on one hand and MS signal intensity on the other.

Development and characterization of cIEF‐MALDI‐TOF MS for protein analysis

“…The third factor in this equation, electrical field strength (E) is also decreased since the same voltage difference is applied over a longer capillary. All three factors are due to a longer capillary and contribute to an increased focusing time compared with the results obtained previously [8]. Overall the optimal focusing time for these experiments was found to be 60-75 min.…”

“…However, these spikes are observed in all UV electropherograms, even at 15 min. Therefore, a different explanation has to be found for protein aggregation or precipitation, since in our previous research spikes were seldom observed with cIEF-UV for this shorter focusing time [8]. In the current setup, however, 37% more sample was injected due to a longer capillary.…”

“…In a previous paper [8] we examined off-line the influence of compounds that are usually added to a cIEF sample to improve repeatability (ampholytes, detergents and viscosity increasing agents) on the signal suppression of proteins in the MALDI-TOF-MS. The compounds providing a relatively high repeatability and resolution in cIEF were selected for these experiments: Pharmalytes, Tween 20 and HEC [8].…”

“…The compounds providing a relatively high repeatability and resolution in cIEF were selected for these experiments: Pharmalytes, Tween 20 and HEC [8]. At deposition of the capillary eluent on the MALDI target plate for 1 min, a spotting time that is often used, it was calculated that 1% w/v of ampholytes reduced the protein MS signal by approximately 50%, while 0.3% HEC decreased the signal intensity by about 60%.…”

“…On the other hand, MALDI is more tolerant to additives like ampholytes and offers the possibility of re-analysis and selection of specific spots for further analysis (MS/MS). Therefore, MALDI is chosen for the experiments described in this paper, although it was shown recently [8] that these additives also significantly decrease the protein signal in MALDI-TOF MS.…”