High‐performance hybrid Orbitrap mass spectrometers for quantitative proteome analysis: Observations and implications

Abstract

We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods on both instruments, namely label-free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3-based reporter ion isolation Synchronous Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label-free approach offers a more linear response with a wider dynamic range than MS/MS-based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We observed, however, that the choice of quantitative approach had little impact on the ability to statistically evaluate the E. coli heat shock response. We conclude that in the experimental conditions tested, MS/MS-based reporter ion quantitation provides reliable biological insight despite the issue of compressed dynamic range, an observation that significantly impacts the choice of instrument.

Alterations in retinoic acid signaling affect the development of the mouse coronary vasculature

“…(Wisniewski et al, 2009; Erde et al, 2014). Tryptic peptides were separated on a nanoACQUITY UPLC analytical column (CSH130 C18, 1.7 μm, 75 μm × 200 mm, Waters) over a 180-minute linear acetonitrile gradient (1–40%) with 0.1 % formic acid on a Waters nano-ACQUITY UPLC system, and analyzed on a coupled Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer as previously described (Williamson et al, 2016). Full scans were acquired at a resolution of 120,000, and precursors were selected for fragmentation by collision-induced dissociation (normalized collision energy at 35%) for a maximum 3-second cycle.…”

Overexpressed Proteins in Hypervirulent Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 Compared to E. coli O157:H7 EDL933 Clade 3 Strain

“…The isobaric tag labeling approach is powerful in providing biologically meaningful clues in comparative pathogen proteomics, when executed on high-performance hybrid orbitrap mass spectrometers [51]. The depth of proteome coverage affects quantitative analysis as well.…”

Towards the Full Realization of 2DE Power

“…Rapid development in ESI LC-MS/MS, especially Orbitrap technology, is accompanied by production of a large amount of data [16]. Here, the combination of 2DE with ESI LC-MS/MS looks like a good choice, since together they allow better representation of the information obtained [88,89].…”

“…Constant improvement of mass spectrometry instruments is expanding the arsenal of data acquisition and analysis approaches. A good example is the Orbitrap series, ESI LC-MS/MS instruments of high mass accuracy (less than 1 ppm), with a high resolution (up to 240,000), and a wide dynamic range (greater than 10 3 ) [15,16]. Despite the high productivity, mass spectrometry in proteomics is still highly dependent on separation technologies that greatly simplify analyses of complex biological samples.…”