Evaluation of front-end higher energy collision-induced dissociation on a benchtop dual-pressure linear ion trap mass spectrometer for shotgun proteomics

Abstract

We report the implementation of front-end higher energy collision-induced dissociation (fHCD) on a benchtop dual-pressure linear ion trap. Software and hardware modifications were employed, described in detail vide-infra, to allow isolated ions to undergo collisions with ambient gas molecules in an intermediate multipole (q00) of the instrument. Results comparing the performance of fHCD and resonance excitation collision-induced dissociation (RE-CID) in terms of injection time, total number of scans, efficiency, mass measurement accuracy (MMA), unique peptide identifications, and spectral quality of labile modified peptides are presented. fHCD is approximately 23% as efficient as RE-CID, and depending on the search algorithm, it identifies 6.6% more or 15% less peptides (q < 0.01) from a soluble whole-cell lysate ( Caenorhabditis elegans ) than RE-CID using Mascot or Sequest search algorithms, respectively. fHCD offers a clear advantage for the analysis of phosphorylated and glycosylated (O-GlcNAc) peptides as the average cross-correlation score (XCorr) for spectra using fHCD was statistically greater (p < 0.05) than for spectra collected using RE-CID.

Figure 1

A schematic of the LTQ Velos Pro (ThermoFisher, San Jose, CA). To implement fHCD, q00 was replaced with an octopole and a DC voltage was placed on the exit lens downstream from the s-lens. Ions are isolated in the ion trap sent back to q00 (70 mtorr) for dissociation and then fragment ions are sent back to the ion trap for subsequent mass analysis.

Figure 2

A) A comparison of the number of ions from the dissociation of MRFA by RE-CID and fHCD. 500 scans were taken at each AGC target value. B) A histogram of the ion injection times from these same experiments. For a given AGC target the IIT were doubled for fHCD to partially account the lower efficiency of fHCD.

Figure 3

The number of unique peptides identified between fHCD and RE-CID from a soluble lysate of C. elegans using Mascot and Sequest. Both searches were post processed with Percolator to enforce a q value of <0.01 at the PSM level. Normal and rapid scan rates were also evaluated (see text for further details).

Figure 4

A heat map and box plot comparing the MMA distribution of fragment ions between A) RE-CID normal scan rate; B) fHCD normal scan rate; C) RE-CID rapid scan rate; D) fHCD rapid scan rate. The heat is the log of the summed fragment ion intensity for each bin. E) A box plot comparing the mass errors of each data set. The line through the box represents the median (50^th percentile) while the box represents the lower (25^th percentile) and upper (75^th percentile) quartiles. Whiskers extend to farthest points that are still 1.5 interquartile ranges within the upper and lower quartiles.

Figure 5

Mass error as a function of number of ions for A) Normal scan RE-CID and B) Normal scan fHCD. The heat is the log of the summed fragment ion intensity for each bin.

Figure 6

A) A comparison between spectra of RE-CID and fHCD (mirrored axis) for the analysis of a phosphorylated peptide. B) fHCD afforded significantly higher spectral quality for this modified peptide as evidenced by the distribution of XCorrs.