On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Abstract

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ~10-s peak width. For 0.1 mg/mL bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4 %) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.

Capillary Isoelectric Focusing-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry for Quantitative Proteomic Analysis of Differentiating PC12 Cells By Eight-Plex Isobaric Tags for Relative and Absolute Quantification

“…3A). This result is likely due to ionization suppression of peptides by the amino acids used as ampholytes [30], which lowers the peptides’ charge. The assumption agreed with the relationship between charge and MH + of peptides from the two approaches, S-Fig.3 in supporting material I.…”

“…We have recently demonstrated that amino acids can act as ampholytes to create the pH gradient in cIEF while generating very low background signals in the m/z range for tryptic peptides [30]. We observed that the background signal intensity generated by the amino acids was a factor of 30 lower than that generated by commercial ampholytes, which was valuable in the study of low abundance peptides by ESI-MS/MS.…”

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

“…Their work is discussed further in the MS detection section of this review. Along with the development of new interfaces, several straightforward BGE buffer modifications have been described in the literature to solve the problems of high backgrounds and ion suppression [63, 64]. …”

Recent developments in capillary and microchip electroseparations of peptides (2011–2013)

“…AAs‐based CIEF of tryptic peptides of BSA with pH gradient formed by six AAs with p I s in the range 3.2–9.7 provided a two‐order lower background signal of ESI‐MS/MS detection than CIEF with commercial polycomponent CAs in the important 300–800 m / z range, resulting in much cleaner mass spectra and higher protein spectral counts. Flattening of pH gradient of CIEF by addition of the particular narrow pH fraction of the CAs to the commercial CAs contributed to the improved resolution of normal and glycated hemoglobins has the potential to enhance also CIEF separation of polypeptides.…”