Combining isoelectric point-based fractionation, liquid chromatography and mass spectrometry to improve peptide detection and protein identification

Abstract

The off-line coupling of an isoelectric trapping device termed membrane separated wells for isoelectric focusing and trapping (MSWIFT) to mass spectrometry-based proteomic studies is described. The MSWIFT is a high capacity, high-throughput, mass spectrometry-compatible isoelectric trapping device that provides isoelectric point (pI)-based separations of complex mixtures of peptides. In MSWIFT, separation and analyte trapping are achieved by migrating the peptide ions through membranes having fixed pH values until the peptide pI is bracketed by the pH values of adjacent membranes. The pH values of the membranes can be tuned, thus affording a high degree of experimental flexibility. Specific advantages of using MSWIFT for sample prefractionation include: (1) small sample volumes (approximately 200 microL), (2) customized membranes over a large pH range, (3) flexibility in the number of desired fractions, (4) membrane compatibility with a variety of solvents systems, and (5) resulting fractions do not require sample cleanup before MS analysis. Here, we demonstrate the utility of MSWIFT for mass spectrometry-based detection of peptides in improving dynamic range and the reduction of ion suppression effects for high-throughput separations of tryptic peptides.

Figure 1

Schematic of MSWIFT assembly including anode/cathode compartments, separation wells and buffering membranes. The four compartment setup was used to separate a mixture of five standard peptides followed by MALDI-MS analysis. The pH values of the buffering membranes used were: 2.9 (A), 5.4 (B), 7.6 (C), 9.0 (D) and 11.0 (E). This configuration would allow for each peptide to be trapped into a single separation well with the exception of angiotensin I & II peptides which are trapped together in the second separation well. A similar configuration was used in all other experiments, except the number of separation wells and the pH values of the buffering membranes were tailored to each separation.

Figure 2

MALDI mass spectrum of a mixture of five peptides before and after IET separation. (A) MALDI mass spectrum of the peptide mixture prior to separation using the MSWIFT device. The mixture contains (a) Bradykinin 1–7 (pI 9.8, [M+H]⁺_obs = 757.39 Da), (b) Angiotensin III (pI 8.8, [M+H]⁺ _obs = 931.54 Da), (c), Angiotensin I (pI 6.9, [M+H]⁺_obs = 1296.75 Da), (d) Angiotensin II (pI 6.7, [M+H]⁺_obs = 1046.58 Da). Each ion signal is labeled with the appropriate peptide with the exception of Leptin (pI 4.4, [M+H]⁺_calc = 1527.81 Da) which is not observed. (B–E) MALDI mass spectra taken from an aliquot of each separation well following IET using MSWIFT. The pH values of the buffering membranes used are indicated in each spectrum. Peptide ion signals are labeled as follows: Bradykinin 1–7 (pI 9.8, [M+H]⁺ _obs = 757.50 Da), Angiotensin III (pI 8.8, [M+H]⁺_obs = 931.66 Da), Angiotensin I (pI 6.9, [M+H]⁺_obs = 1296.88 Da), Angiotensin II (pI 6.7, [M+H]⁺_obs = 1046.68 Da), Leptin (pI 4.4, [M+H]⁺_obs = 1527.99 Da).

Figure 3

(A–F). MALDI mass spectrum of the contents of each MSWIFT separation well from the IET separation of the five protein digestion mixture. The mixture includes tryptic peptides from the proteins bovine serum albumin, apo-transferrin, ribonuclease A, α_s1-casein, and cytochrome c. The pH values of the buffering membranes used were as follows: (1) pH 2.0–4.5, (2) pH 4.5–5.4, (3) pH 5.4–6.5, (4) pH 6.5–7.6, (5) pH 7.6–8.2 and (6) pH 8.2–12.0. Peptide ion signals denoted with a filled circle (●) are those which are not observed in the mass spectrum acquired prior to IET separation.

Figure 4

Plot of the number of unique proteins identified in each fraction of the MSWIFT. The final column represents the total number of proteins identified by database searching tandem MS data obtained from all six fractions.