Evaluating the Characteristics of Reporter Ion Signal Acquired in the Orbitrap Analyzer for Isobaric Mass Tag Proteome Quantification Experiments

Abstract

Multiplexed quantification with isobaric chemical tags (e.g., TMT, iTRAQ) provides a robust and efficient means to comparatively examine proteome dynamics between several biological states using a mass spectrometer (MS). The quantitative nature of isobaric tags necessitates strict validation of the observed ion signals in the chosen MS detector before differential patterns are extracted between biological states. We present an in-depth analysis of isobaric tag data acquired on current generation Orbitrap MS hardware to illustrate pitfalls in acquisition settings that can negatively impact results. We establish, for the first time, the presence of a notch, a region of no observed values, in the reporter ion distributions from isobaric-labeled peptide mixtures acquired on these instruments. We determine that this notch is present in published data across a wide range of instruments of the same or different type and is isolated to the Orbitrap mass analyzer. We demonstrate that the impact of the notch can be minimized using manipulations of Orbitrap scan parameters and on-column injection amounts. Lastly, using a mixture of synthetic standard peptides we investigated the impact on identification rates and quantification precision. Together, these data highlight an important phenomenon that negatively impacts peptide identification and quantification in the Orbitrap analyzer as well as outlining guidelines to follow to ensure minimization of MS-induced artifacts in isobaric tag experiments resulting from the notch.

Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

“…Furthermore, we should reject TMT-peptides without reporter ion signals in the sample channels. Therefore, we examined the distribution of the TMT reporter ion intensity of each sample channel and found a notch to discriminate the signal from the noise (Figure S5); this was also mentioned in the previous study (Hughes et al, 2017). Based on this observation, we set the acceptance criterion for the minimum TMT intensity in the sample channels to be greater than 40 on a log2 scale for the total TMT intensity of the sample channels.…”

Prior Signal Acquisition Software Versions for Orbitrap Underestimate Low Isobaric Mass Tag Intensities, Without Detriment to Differential Abundance Experiments

“…Thus, we generated differences in protein abundance between 1.06 -6-fold for H.sapiens and S.cerevisiae proteins. Peptides were then labeled with TMT and quantified on an Orbitrap Mass Spectrometer with Tune 2.1 and Tune 3.4, with an AGC target of 50,000 and max injection time of 120 ms, which we expected to yield a moderate and typical notch 9 (see methods for details). Using these parameters and Tune 2.1, 3.2 % of tag intensities were within or below the notch (Figure 2b), with S.cerevisiae peptides showing a slightly greater proportion of low tag intensities (Figure S3b-c).…”

“…Thus, we generated differences in protein abundance between 1.06 -6-fold for H.sapiens and S.cerevisiae proteins. Peptides were then labeled with TMT and quantified on an Orbitrap Mass Spectrometer with Tune 2.1 and Tune 3.4, with an AGC target of 50,000 and max injection time of 120 ms, which we expected to yield a moderate and typical notch 9 (see methods for details). Using these parameters and We applied strict filtering to minimise the possibility of PSM mis-identification or interference, demanding co-isolation < 10 %, and delta score > 0.5, with the later ensuring that rank 2 peptide matches for a given spectrum have a score less than half the rank 1 peptide match.…”

“…Hughes et al performed a spike-in benchmark experiment to assess the impact of the notch 9 . However, this included only 550 spike-in peptides, precluding a consideration of the impact of the notch in a routine proteomics experiment.…”

“…Thus, robust quantification of TMT labelled peptides requires highresolution, accurate mass spectrometers, with Orbitrap™ devices being commonly employed. Intriguingly, a recent characterisation of TMT reporter ion signals obtained from Orbitrap identified a consistent absence of intensities within a specific range, which visually appears as 'notch' in the distribution of intensities 9 . The presence of the notch was determined to depend on the automatic gain control (AGC) target and maximum injection time Orbitrap parameters, with higher values reducing its prominence.…”