As a driver for many biological processes phosphorylation remains an area of intense research interest. of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion Fostamatinib disodium intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan (iii) interfering ions generally possessed a different charge state than the target precursor and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent low level background fragmentation. Finally as a proof-of-concept 10-plex experiment we compared phosphopeptide levels from five murine brains to five livers. In total the SPS-MS3 method quantified 38?247 phosphopeptides corresponding to 11?000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide this equates to Fostamatinib disodium more than 628?000 binary comparisons collected in less than 48 h. As a key mediator of cellular signaling phosphorylation remains a principal target for biological interrogation.1 Identifying and quantifying the phosphorylation state of proteins involved in cell progression metabolism growth and disease is critical for the continued elucidation of cellular function.2 Global phosphoproteome characterization is challenging due to the estimated large volume of phosphorylation sites in eukaryotic cells and the often low abundance/stoichiometry of the phosphoproteome.3 4 Continuing technological and methodological advancements have resulted in the characterization of tens of thousands of phosphorylation sites across numerous species but it is apparent that only a fraction of all phosphorylation events have been characterized.5?11 Furthermore phosphorylation dynamics assessed via relative quantification have historically been limited to binary or ternary comparisons further limiting the breadth and depth of phosphopeptide analysis.12?17 Novel methodologies are needed in order to overcome the current shortcomings of phosphoproteome characterization. Mass spectrometry remains an unmatched platform for comprehensive phosphoproteome analysis. Coupling deep identification with relative quantification has provided valuable biological insights that would be otherwise unobtainable by traditional biochemical techniques.18?24 Isobaric tags for relative and absolute quantitation (iTRAQ) and tandem-mass-tag (TMT) based methodologies permit the simultaneous comparison of up to 8 Fostamatinib disodium (iTRAQ) or 10 (TMT) samples facilitating complex experimental designs and the inclusion of biological replicates within the same experiment. A primary hurdle for isobaric based quantification technologies is the presence of interfering coisolated species that result in distorted reporter ion intensities. A number of publications have documented this phenomenon and several have demonstrated approaches to alleviate the interference.25?31 One such approach was the inclusion of a quantitative MS3 spectrum.32 Recently the sensitivity of the MS3 method was dramatically improved by isolating multiple fragment ions in the MS2 spectrum using isolation waveforms with multiple notches (e.g. synchronous precursor selection SPS).33 The SPS-MS3 method is available on the Orbitrap Fusion Fostamatinib disodium which leverages advancements in software and hardware to provide increased scan rates and improved sensitivity resolution and quantitative accuracy. Furthermore a unique architecture expands the concept of a hybrid mass spectrometer by incorporating three mass analyzers (i.e. quadrupole mass filter quadrupole ion trap and Orbitrap) operating in a task parallelized manner. Here we IP1 assessed the performance of the SPS-MS3 method on two different phosphoproteome samples. We utilized a 2-phosphoproteome model of interference to characterize the quantitative accuracy of various SPS-MS3 and MS2 methods Fostamatinib disodium on the Orbitrap Fusion. We observed that known ratios were distorted for the MS2 method compared to the SPS-MS3 method. In a large-scale demonstration of the method we performed a proteome-wide.