A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments

Abstract

Isobaric labeling is a powerful strategy for quantitative mass spectrometry-based proteomic investigations. A complication of such analyses has been the co-isolation of multiple analytes of similar mass-to-charge resulting in the distortion of relative protein abundance measurements across samples. When properly implemented, triple-stage mass spectrometry and synchronous precursor selection (SPS-MS3) can reduce the occurrence of this phenomena, referred to as ion interference. However, no diagnostic tool is available currently to rapidly and accurately assess ion interference. To address this need, we developed a multiplexed TMT-based standard, termed the triple knockout (TKO). This standard is comprised of three yeast proteomes in triplicate, each from a strain deficient in a highly abundant protein (Met6, Pfk2, or Ura2). The relative abundance patterns of these proteins, which can be inferred from dozens of peptide measurements, are representative of ion interference in peptide quantification. We expect no signal in channels where the protein is knocked out, permitting maximum sensitivity for measurements of ion interference against a null background. Here, we emphasize the need to investigate further ion interference-generated ratio distortion and promote the TKO standard as a tool to investigate such issues.

Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling

“…Afterwards, biotinylated proteins were enriched by denaturing streptavidin affinity purification, digested into tryptic peptides, labeled with isobaric tandem mass tags (TMT), fractionated by alkaline reversed phase chromatography, and analyzed by triple stage mass spectrometry (TMT SPS MS 3 ; Figure 1B). Quantification by SPS MS 3 rather than applying conventional tandem mass spectrometry (MS 2 ) has the advantage of reducing signal ratio distortion and enhancing the accuracy of peptide (and by inference, protein) quantification (McAlister et al, 2014; Paulo et al, 2016b; Ting et al, 2011). …”

“…Proteins were quantified by summing reporter ion counts across all matching PSM. Data tables (Table S1–3) were generated requiring an MS 2 isolation specificity of >70% for each peptide and a sum of TMT s/n of >100 across all channels for any given peptide and exported to Excel and further processed therein (Paulo et al, 2016b). A modified version of the Ascore algorithm was used to quantify the confidence assignment of phosphorylation sites.…”

Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription

“…Peptides were separated using a 2 h gradient of 7 to 27% acetonitrile in 0.125% formic acid at a flow rate of ~450 nL/min. Each analysis used the multi-notch MS3-based TMT method 34 on an Orbitrap Fusion mass spectrometer. The scan sequence began with an MS1 spectrum (Orbitrap analysis; resolution 120,000; mass range 400–1,400 m/z; automatic gain control (AGC) target 5 × 10 5 ; maximum injection time 100 ms).…”

Multiplexed Isobaric Tag‐Based Profiling of Seven Murine Tissues Following In Vivo Nicotine Treatment Using a Minimalistic Proteomics Strategy

“…For each analysis, we loaded ~2 μg onto the column and separation was achieved using a 2.5 h gradient of 7 to 27% acetonitrile in 0.125% formic acid at a flow rate of ~550 nL/min. Each analysis used an SPS-MS3-based TMT method [9, 10] , which has been shown to reduce ion interference compared to MS2-based quantification [11] . The scan sequence began with an MS1 spectrum (Orbitrap; resolution 120,000; mass range 400–1400 m/z; automatic gain control (AGC) target 5 × 10 5 ; maximum injection time 100 ms).…”