Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes

Abstract

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.

Fig. 1.

The Orbitrap Elite mass spectrometer. A, Novel elements compared with the LTQ Orbitrap Velos are highlighted and encompass the source region, the dual linear ion trap and the Orbitrap analyzer. ETD fragmentation is optional. B, Computer model of the inlet ion optics, showing the S-lens on the left and the dual ion trap on the right. The bent, square transfer quadrupole allows neutrals to leave the ion optics and impinge on the depicted beam blocker. C, Comparison of dimensions of the standard (left) to the compact, high-field Orbitrap analyzer (right).

Fig. 2.

Top down method at an LC time scale. Total ion chromatogram of an LC separation of carbonic anhydrase II (29 kDa). Fast survey scans reveal the charge envelope and are followed by high resolution SIM and HCD scans. Overlapping fragment isotope distributions are clearly resolved from each other.

Fig. 3.

Isotope resolved spectrum of enolase with Orbitrap Elite and LTQ Orbitrap Velos instruments. Spectra were acquired with transients of A, 768 ms on the Orbitrap Elite and B, 1536 ms on the LTQ Orbitrap Velos. In these conditions, the superior resolution of the Orbitrap Elite instrument (>2 × higher in 2 × shorter transient time) helps to baseline resolve the 47+ charge state of the intact yeast enolase.

Fig. 4.

Parallel CID top20 method and ultra high resolution survey scans. A, High resolution MS scan at three different transient lengths followed by 20 CID MS/MS scans in the linear ion trap. Note that cycle time is unaffected by the resolution of the full scan. Preview refers to the portion of the survey scan that is used to select precursor ions for fragmentation. B, LC MS heat map of peptides eluting over a 3 min elution time interval in a 40 Th range. More detail is visible in the ultra high resolution setting (left panel) compared with the normal resolution setting (right panel). C, Separation of isobaric species in standard LC MS/MS analysis. The ³⁴S isotope containing peak is clearly resolved from the ¹³C₂ isotope.

Fig. 5.

Combinations of fragmentation modes. A, Sequential mode in which a high resolution survey scan with 120,000 resolution (384 ms) is followed by 15 HCD scans at 15,000 resolution (48 ms transients). B, Parallel and sequential mode in which an ultra high survey scan with 240,000 resolution is acquired in parallel with 10 rCID spectra in the linear ion trap and in sequence with 10 HCD scans that are analyzed in the Orbitrap analyzer. The same precursors are analyzed by rCID and HCD. C, Double sequential mode in which a 120,000 resolution survey scan is followed by 5 high resolution CID and 5 HCD spectra of the same precursors.

Fig. 6.

Complementary CID + HCD MS/MS spectra. Example spectra using the parallel and sequential fragmentation mode depicted in Fig. 5B. A, The rCID spectrum features more b-ions than the HCD spectrum. B, HCD spectrum with nearly complete y-ion series. Mass accuracy in B but not in A is in the ppm range (absolute average deviation of 3.67 ppm. versus 0.06 Th).