Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry

Abstract

Advancement in proteomics research relies on the development of new, innovative tools for identifying and characterizing proteins. Here, we describe a protocol for analyzing peptides and proteins on a chromatographic timescale by coupling nanoflow reverse-phase (RP) liquid chromatography (LC) to electron-transfer dissociation (ETD) mass spectrometry. For this protocol, proteins can be proteolytically digested before ETD analysis, although digestion is not necessary for all applications. Proteins <or=30 kDa can be analyzed intact, particularly if the objective is protein identification. Peptides or proteins are loaded onto a RP column and are gradient-eluted into an ETD-enabled mass spectrometer. ETD tandem mass spectrometry (MS/MS) provides extensive sequence information required for the unambiguous identification of peptides and proteins and for characterization of posttranslational modifications. ETD is a powerful MS/MS technique and does not compromise the sensitivity and speed necessary for online chromatographic separations. The described procedure for sample preparation, column packing, sample loading and ETD analysis can be implemented in 5-15 h.

Figure 1

Fragmentation scheme for production of ions of type c′ and z′^• resulting from the reaction of a fluoranthene radical anion and a multiply protonated peptide,. c′- and z′^•-type product ions are generated following transfer of a low-energy electron from fluoranthene to a carbonyl group solvated to the side chain of a basic residue, such as lysine.

Figure 2

ETD mass spectrum of the [M + 3H]⁺³ ion (m/z 519.8) of a phosphorylated peptide derived from the HIV-1 Rev protein. Singly and doubly charged product ions of type c′ and z′^• are listed above and below the peptide sequence, and the observed ions are underlined and labeled in the MS/MS spectrum. Singly and doubly charged product ion masses were calculated as monoisotopic and average masses, respectively, and are listed with no decimal places. represents a phosphate group and is positioned above the phosphorylated serine residue. A triangle (▼) is positioned above m/z peaks that are within the precursor isolation window. Charge-reduced species and species resulting from neutral losses are bracketed.

Figure 3

Diagram of a capillary column utilized for online nanoflow LC analyses. Irregular sized RP resin (A) is contained in a length of fused silica capillary tubing by a LiChrosorb frit (B). An analytical column is attached to the precolumn using teflon tubing (C). The analytical column is packed with regular-sized RP resin (D). The regular-sized resin is retained in the column with a small amount of irregular RP resin (E) and a bottleneck restriction (F). Capillary columns are equipped with an integrated electrospray emitter tip (G).

Figure 4

ETD mass spectra recorded, with and without supplemental activation, on [M + 3H]⁺³ ions (m/z 823) from residues 18–39 of Adrenocorticotropic Hormone (ACTH 18–39). (a) ETD spectrum (reaction time, 400 ms) dominated by the charge-reduced species [M + 3H]^+2• at m/z 1,233. (b) and (c) Same ETD spectrum, displayed in 1 and 3-panel views, respectively, acquired after supplemental activation was applied to the charge-reduced [M + 3H]^+2• and [M + 3H]^+1•• ions at m/z 1,233 and 2,466. (d) Predicted m/z values for singly and doubly charged fragment ions of type c′ and z′^• displayed above and below the amino acid sequence of ACTH (18–39). Ions observed in the spectrum are underlined. Note that hydrogen radical transfer from c′- to z′^•-type ions in the long-lived, charge-reduced species can shift the observed signals by minus and plus 1 Da, respectively.

Figure 5

ETD/PTR mass spectrum of a 90-residue peptide derived from the Sam68 protein. The ion selected for dissociation was the [M + 13H]⁺¹³ ion (m/z 699.4). ETD and PTR times were 40 ms and 135 ms, respectively. The c′- and z′^•-type product ions (singly and doubly charged) that occur from cleavage at the N- and C-terminal 30 residues of the peptide are listed above and below the sequence, respectively. Note that cleavage N-terminal to proline residues does not occur under ETD conditions. Observed singly and doubly charged product ions are underlined and labeled in the MS/MS spectrum. is positioned above the arginine residue found to be dimethylated. A triangle (▼) is positioned above m/z peaks that are within the precursor isolation window.

Figure 6

ETD/PTR mass spectrum of intact 21-kDa protein, p21. The ion selected for dissociation was the [M + 24H]⁺²⁴ ion (m/z 851.6). ETD and PTR times were 40 and 135 ms, respectively. The first (N-terminal) and last (C-terminal) 30 residues are shown, whereas the remaining 117 residues of the protein are not shown. The c′- and z′^•-type product ions that occur from cleavage at the N- and C-terminal 30 residues of the peptide are listed above and below the sequence, respectively. Note than cleavage N-terminal to proline residues does not occur under ETD conditions. Observed singly and doubly charged product ions are underlined and labeled in the MS/MS spectrum.