Comparison of the LTQ-Orbitrap Velos and the Q-Exactive for proteomic analysis of 1-1000 ng RAW 264.7 cell lysate digests.

RATIONALE There is interest in extending bottom-up proteomics to the smallest possible sample size. We investigated the performance of two modern mass spectrometers for the analysis of samples ranging from 1 ng to 1 µg of RAW 264.7 cell lysate digests. METHODS An ultra-performance liquid chromatography (UPLC) system coupled with either an LTQ-Orbitrap Velos or a Q-Exactive mass spectrometer was used for peptide separation and identification. RESULTS For 1-1000 ng RAW 264.7 cell lysate digests, the Q-Exactive generated 10-83% more protein groups and 11-109% more peptides than the LTQ-Orbitrap Velos (higher-energy collisional dissociation, HCD) with MASCOT database searching, due to its faster scan rate and higher resolution. In addition, HCD and collision-induced dissociation (CID) modes of the LTQ-Orbitrap Velos were compared. HCD produced higher peptide and protein group IDs than CID for 1-1000 ng RAW 264.7 cell lysate digests with MASCOT database searching. Database searching results from SEQUEST and MASCOT were also compared and comparable protein group IDs were obtained from the two search engines. CONCLUSIONS The Q-Exactive outperformed the LTQ-Orbitrap Velos for shotgun proteomics analysis of 1 to 1000 ng RAW 264.7 cell lysate digests in terms of obtained peptide and protein group IDs.

[1]  Edward L. Huttlin,et al.  Evaluation of HCD- and CID-type Fragmentation Within Their Respective Detection Platforms For Murine Phosphoproteomics* , 2011, Molecular & Cellular Proteomics.

[2]  R. Aebersold,et al.  Mass Spectrometry and Protein Analysis , 2006, Science.

[3]  Richard J. Lavallee,et al.  Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer. , 2012, Journal of proteome research.

[4]  N. Dovichi,et al.  High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres. , 2012, Journal of chromatography. A.

[5]  M. Mann,et al.  Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer* , 2011, Molecular & Cellular Proteomics.

[6]  M. Mann,et al.  Deep and Highly Sensitive Proteome Coverage by LC-MS/MS Without Prefractionation* , 2011, Molecular & Cellular Proteomics.

[7]  M. Mann,et al.  System-wide Perturbation Analysis with Nearly Complete Coverage of the Yeast Proteome by Single-shot Ultra HPLC Runs on a Bench Top Orbitrap* , 2011, Molecular & Cellular Proteomics.

[8]  Ronald J. Moore,et al.  Effectiveness of CID, HCD, and ETD with FT MS/MS for degradomic-peptidomic analysis: comparison of peptide identification methods. , 2011, Journal of proteome research.

[9]  N. Dovichi,et al.  CZE‐ESI‐MS/MS system for analysis of subnanogram amounts of tryptic digests of a cellular homogenate , 2012, Proteomics.

[10]  M. Mann,et al.  Precision proteomics: The case for high resolution and high mass accuracy , 2008, Proceedings of the National Academy of Sciences.

[11]  Richard D. Smith,et al.  Improving collision induced dissociation (CID), high energy collision dissociation (HCD), and electron transfer dissociation (ETD) fourier transform MS/MS degradome-peptidome identifications using high accuracy mass information. , 2012, Journal of proteome research.

[12]  Matthias Mann,et al.  A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed* , 2009, Molecular & Cellular Proteomics.

[13]  M. Mann,et al.  Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation. , 2010, Journal of proteome research.

[14]  S. Mohammed,et al.  Improved peptide identification by targeted fragmentation using CID, HCD and ETD on an LTQ-Orbitrap Velos. , 2011, Journal of proteome research.