/ mass spectrometry

Introducing a new lab member, the Q-Exactive HF-X

Unboxing a new instrument is always exciting, but the Q-Exactive HF-X probably represents the most significant upgrade to the Q-Exactive line since it was launched in 2011.[1] Q-Exactives are quadrupole-Orbitrap hybrid mass spectrometers. The Orbitrap mass analyzer enables the very high mass resolution and mass accuracy measurements which are characteristic of this instrument platform. The Orbitrap line has steadily improved,[2] most notably in sequencing acquisition speed and sensitivity.

The HF-X can profile over 2,500 proteins, in 1/6 the time, with 1/5 the sample, as the original Q-Exactive. [3] That's over 2,500 proteins in 15 minutes from 1 microgram of peptide digest, and 4,000 proteins in 60 mintues. The HF-X can profile to a similar depth as the previous generation, the Q-Exactive HF, using 1/10 the sample amount - 100 nanograms.

In internal testing, we found, consistent with the published results, that the HF-X could profile 2,800 proteins from 1 microgram of HeLa digest in a 15 minute UPLC gradient; 4,300 proteins from 1 microgram of HeLa digest in a 60 minute UPLC gradient; and 2,000 proteins from 100 nanograms of HeLa digest in a 15 minute UPLC gradient.

Another interesting detail from the paper is that the HF-X can sequence at up to over 1,000 unique peptides per minute. The "unique" qualification is important. It would not be very useful if the instrument sequenced the same albumin peptides in your plasma digest over and over again. Or sequenced what turned out to be noise peaks in a trace sample. To generate a useful proteomic profile the instrument needs to determine which peaks to isolate for fragmentation and sequencing, which to ignore and which it has already acquired. The HF-X implements a new algorithm, "advanced peak determination," (APD) to accomplish this. [4] To oversimplify, APD improves the ability of the instrument to determine what is, and what is not, a peptide ion, so that the tandem MS acquisitions are performed more often on peptides and less often on noise. This results in an improvement of sampling capacity from 72% to 96%, relative to the algorithm used by previous generation instruments. The improvement in peptide feature detection also benefits the match-between-run algorithm used in various data processing platforms such as MaxQuant and OpenMS. Because more peptide features are annotated, more features can be matched across sample runs in an experiment.

These dramatic improvements in speed and sensitivity help to address longstanding issues with mass spectrometry-based proteomics: the requirement for larger sample amounts than were sometimes available, especially for clinical samples, and for costly, time consuming experiments. The Q-Exactive HF-X allows us to offer you cost-effective proteomic profiling of thousands of proteins, from as little as 100 nanograms of protein, in minutes.


  1. Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer ↩︎

  2. Evolution of Orbitrap Mass Spectrometry Instrumentation ↩︎

  3. Performance evaluation of the Q Exactive HF-X for shotgun proteomics ↩︎

  4. Improved Precursor Characterization for Data-Dependent Mass Spectrometry ↩︎