In-capillary enrichment, proteolysis and separation using capillary electrophoresis with discontinuous buffers: application on proteins with moderately acidic and basic isoelectric points

Abstract

Advances in mass spectrometry and capillary-format separation continue to improve the sensitivity of protein analysis. Of equal importance is the miniaturization of sample pretreatment such as enrichment and proteolysis. In a previous report (Nesbitt et al., Electrophoresis, 2008, 29, 466-474), nanoliter-volume protein enrichment, tryptic digestion, and partial separation was demonstrated in capillary electrophoresis followed by MALDI mass spectral analysis. A discontinuous buffer system, consisting of ammonium (pH 10) and acetate (pH 4), was used to create a pH junction inside the capillary, trapping a protein with a neutral isoelectric point, myoglobin (pI 7.2). Moreover, co-enrichment of myoglobin with trypsin led to an in-capillary digestion. In this paper, the ability of this discontinuous buffer system to perform similar in-capillary sample pretreatment on proteins with moderately acidic and basic pI was studied and reported. Lentil lectin (pI 8.6) and a multi-phosphorylated protein, beta-casein (pI 5.1), were selected as model proteins. In addition to the previously shown tryptic digestion, proteolysis with endoproteinase Asp-N was also performed. Digestion of these acidic and basic pI proteins produced a few peptides with extreme pI values lying outside the trapping range of the discontinuous buffer. An alteration in the peptide trapping procedure was made to accommodate these analytes. Offline MALDI mass spectral analysis confirmed the presence of the expected peptides. The presented miniaturized sample pretreatment methodology was proven to be applicable on proteins with a moderately wide range of pI. Flexibility in the choice of protease was also evident.

Removal of sample background buffering ions and myoglobin enrichment via a pH junction created by discontinuous buffers in capillary electrophoresis

“…Myoglobin is selected as the model protein in this work due to its unique UV absorption at 408 nm for identification. The enrichment of other proteins (carbonic anhydrase I, bovine serum albumin, lentil lectin, and ␤-casein) and peptides (tryptic digests of myoglobin, lentil lectin and ␤-casein, and endoproteinase Asp-N digest of myoglobin) with the same discontinuous buffer system has been demonstrated in previous reports [32,35,36].…”

“…Enrichment factors of up to 2000 were reported. Following the enrichment, the stacked protein or protein mixture molecules were subsequently separated using capillary zone electrophoresis (CZE) [34], spotted onto a MALDI target for MS analysis [35], or digested into peptides with trypsin prior to MS analysis [35,36].…”

Study of the dynamical behavior of sodium alginate/myoglobin aqueous solutions: A dynamic light scattering study

“…Nevertheless, according to recent studies on the same Sigma product [25,26], a value of 7.2 can be considered as a good estimation of the myoglobin pI. For this reason we refer to this value for our following considerations.…”

Synthesis of coumarin-appended cyclophanes and evaluation of their complexation with myoglobin

“…Electrostatic interactions between the hosts and myoglobin were not effectively performed, because the K value of anionic host 1b with myoglobin was almost the same with that of cationic host 1a with myoglobin: K, 1.5 9 10 5 M -1 for 1b with myoglobin. According to the literature [31], isoelectric point of myoglobin is 7.2. Under the experimental condition of pH 7.4, myoglobin maintains almost balance of positive and negative charges on its surface.…”