Capillary isoelectric focusing coupled offline to matrix assisted laser desorption/ionization mass spectrometry

Abstract

This work presents several critical details for making cIEF-MALDI-MS a robust technique which will allow for more routine application and aid in automation. This includes emphasis on the hardware necessary for syringe pump mobilization and proper protocol for preventing disruption from gas bubbles. Following these guidelines, excellent elution time reproducibility is demonstrated for six pI markers (RSD < 5%). Additionally, the pI markers are used to calibrate the pH gradient and determine experimental pIs of proteins detected offline by mass spectrometry. This was demonstrated using a standard protein mixture of myoglobin and two forms of β-lactoglobulin. Experimental determination of protein pIs and molecular weights were found to be in agreement with literature values. The technical details discussed provide a sound foundation for applying the offline coupling of MALDI-MS with cIEF.

Isoelectric Focusing in a Drop

“…Myoglobin (pI 7.2) was selected as a target since it has a chromophore allowing easy detection and its IEF properties were previously studied 39. Assuming a linear pH gradient and defining the anode as pH 3 at x =0 and the cathode as pH 10 at x =1.5 cm means that myoglobin would focus at x pI =0.9 cm.…”

“…Previously, myoglobin focused into a band with a standard deviation of 0.6 cm in a 40 cm capillary while applying 10 kV 39. Thus, when the separation length is 40 cm then α =0.01 V −1 cm −1 for myoglobin.…”

PI-Based Separation of Tissue Proteins Realized with the Use of Two-Dimensional Gel Electrophoresis or Alternatively Capillary Isoelectric Focusing

“…In cIEF, carrier ampholyte mixtures are used to establish a stable pH gradient inside a capillary suspended between an acid and base reservoir. Amphoteric analytes, typically proteins, will migrate with their local electrophoretic mobility until reaching the pH where their net charge is zero, defined as the isoelectric point (pI) [17].…”

Recent developments in electrophoretic separations on microfluidic devices

“…Several groups continue to develop variations on standard electrophoretic separation techniques, including Astorga-Wells [14], Gebauer [15,16], Hayes [17,18], and Ivory [19,20], but the current article will center on elements that are essentially new or are significant advances in strategies that uniquely exploit microfluidic formats. The advantages of microfluidic devices include lower sample consumption, portability, and shorter analysis times.…”