Continued advancements in instrumentation, sample preparation methodologies and bioinformatics1–2
have increased the utility of mass spectrometry to address complex biological questions in the last decade. On the instrumentation front, the field has witnessed significant improvements including the development of hybrid FT-MS instruments3–4
, the introduction of the orbitrap mass analyzer5–6
, and improvements in time-of-flight (TOF) technology7–8
. These developments have led to unprecedented instrument performance in terms of resolving power, mass measurement accuracy, dynamic range, and duty cycle. In combination with these advancements, new dissociation techniques including electron capture dissociation9
(ECD) and electron transfer dissociation10
(ETD) continue to drive the field of proteomics forward. ECD and ETD offer complimentary sequence information to collision induced dissociation (CID) and thus are becoming more widely used. In addition, due to the nonergodic nature of ECD and ETD, they are often the preferred method for pinpointing sites of labile modifications (e.g., phosphorylation, O-GlcNAc) along the peptide backbone.
The traditional method for peptide fragmentation, CID11
, is a slow heating technique12
that uses multiple gas collisions to increase internal energy through vibrational pathways to an ion specific threshold prior to uni-molecular dissociation. It is a common method to obtain structural information, possibly owing to its relative simplicity and high efficiency, and is employed on a range of mass spectrometry platforms in slightly different varieties. Traditionally, CID has been accomplished using beam-type or tandem in space instruments (e.g., Triple quadrupole, q-TOF) in which selected ions are accelerated into an elevated pressure cell and subjected to collisions with inert gas molecules. For quadrupole ion trap and FT-ICR instruments (i.e., tandem in time), CID is achieved through resonance excitation collision induced dissociation (RE-CID) or sustained off resonance irradiation collision induced dissociation (SORI-CID)13
, respectively. SORI-CID has seen limited use in the field of shotgun proteomics – largely due to the development of hybrid instruments and lengthy pump down periods associated with the method which are not realistic on an LC-MS timescale. A disadvantage of RE-CID for proteomic applications is the inherent low mass cut off which is a consequence of the magnitude of the radio frequency voltage needed to retain the resonantly excited precursor and resulting fragments. In beam-type CID the low mass ions are retained for mass analysis and as a result this technique is directly amenable to isobaric tagging strategies (iTRAQ)14
for multiplexed relative quantification. In addition, due the tendency for multiple bond breakage in beam-type dissociation as opposed to single bond breakage in RE-CID, immonium ions are often present and can be utilized to obtain sequence information.15
Mann and coworkers described beam-type fragmentation on a hybrid ion trap orbitrap in the c-trap or dedicated octopole and referred to the method as higher energy collision-induced dissociation (HCD).16
The latter version of the instrument is now commercially available on the orbitrap line of instruments (Thermo Fisher, San Jose, CA). To the authors’ knowledge this was the first report of beam-type fragmentation on a trapping-type instrument; however, implementation of HCD on a benchtop trapping instrument would also be beneficial for similar reasons described vide supra
– in addition to the lower cost associated with the purchase and operation of these instruments. Recently, Coon and coworkers reported HCD capabilities on a benchtop quadrupole linear ion trap17
(LTQ XL, Thermo Fisher Scientific, San Jose, CA). In this work the instrument software was modified to enable isolated ions to be sent back through the high pressure regions of the ion trap to q0, colliding with background ambient gas molecules, and then fragments were sent back to the ion trap for mass spectral analysis. This study focused on optimizing instrument parameters for HCD (e.g., offset voltages, ejection times and collision energies) and illustrated an increase in quantifiable peptides using trap HCD compared to other dissociation techniques in an iTRAQ experiment.
Herein, we describe the capability to perform front-end higher energy collision induced dissociation (fHCD, commercially called “Trap HCD”) on a bench top dual pressure linear ion trap (LTQ-Velos Pro, Thermo Fisher Scientific, San Jose, CA). It is important to distinguish the setup described herein with the one reported by Coon and coworkers. A design characteristic, inherent to the Velos ion trap instruments, prevents beam-type fragmentation in the q0 region of the instrument due to the inability to accelerate ions of appreciable energy through a curved quadrupole (i.e., q0). In our setup, isolated ions are sent back to q00 multipole downstream from the S-lens, for beam-type fragmentation and fragments are then mass analyzed by the ion trap. A distinct advantage of this setup is the small length of q00 (25 mm) versus q0 (85 mm) which affords approximately a 10-fold decrease in extraction time. Ultimately, this decrease in extraction time yields an overall increase in duty cycle. In addition, the instrument reported here is commercially available from ThermoFisher (as the LTQ-Velos Pro).
The manuscript is divided into three separate experiments: 1) Determination of the efficiency of fHCD in relation to RE-CID; 2) A detailed comparison of the performance of fHCD and RE-CID for shotgun proteomics in terms of unique peptide identifications, total number of scans, and mass measurement accuracy; and 3) A comparison between fHCD and RE-CID for the analysis of peptides with labile modifications.