FAIMS Enhances the Detection of PTM Crosstalk Sites

Protein post-translational modifications (PTMs) enable cells to rapidly change in response to biological stimuli. With hundreds of different PTMs, understanding these control mechanisms is complex. To date, efforts have focused on investigating the effect of a single PTM on protein function. Yet, many proteins contain multiple PTMs. Moreover, one PTM can alter the prevalence of another, a phenomenon termed PTM crosstalk. Understanding PTM crosstalk is critical; however, its detection is challenging since PTMs occur substoichiometrically. Here, we develop an enrichment-free, label-free proteomics method that utilizes high-field asymmetric ion mobility spectrometry (FAIMS) to enhance the detection of PTM crosstalk. We show that by searching for multiple combinations of dynamic PTMs on peptide sequences, a 6-fold increase in candidate PTM crosstalk sites is identified compared with that of standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflows. Additionally, by cycling through FAIMS compensation voltages within a single LC-FAIMS-MS/MS run, we show that our LC-FAIMS-MS/MS workflow can increase multi-PTM-containing peptide identifications without additional increases in run times. With 159 novel candidate crosstalk sites identified, we envisage LC-FAIMS-MS/MS to play an important role in expanding the repertoire of multi-PTM identifications. Moreover, it is only by detecting PTM crosstalk that we can “see” the full picture of how proteins are regulated

FAIMS Enhances the Detection of PTM Crosstalk Sites

Protein post-translational modifications (PTMs) enable cells to rapidly change in response to biological stimuli. With hundreds of different PTMs, understanding these control mechanisms is complex. To date, efforts have focused on investigating the effect of a single PTM on protein function. Yet, many proteins contain multiple PTMs. Moreover, one PTM can alter the prevalence of another, a phenomenon termed PTM crosstalk. Understanding PTM crosstalk is critical; however, its detection is challenging since PTMs occur substoichiometrically. Here, we develop an enrichment-free, label-free proteomics method that utilizes high-field asymmetric ion mobility spectrometry (FAIMS) to enhance the detection of PTM crosstalk. We show that by searching for multiple combinations of dynamic PTMs on peptide sequences, a 6-fold increase in candidate PTM crosstalk sites is identified compared with that of standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflows. Additionally, by cycling through FAIMS compensation voltages within a single LC-FAIMS-MS/MS run, we show that our LC-FAIMS-MS/MS workflow can increase multi-PTM-containing peptide identifications without additional increases in run times. With 159 novel candidate crosstalk sites identified, we envisage LC-FAIMS-MS/MS to play an important role in expanding the repertoire of multi-PTM identifications. Moreover, it is only by detecting PTM crosstalk that we can “see” the full picture of how proteins are regulated

Differential Phosphoproteomics of Fibroblast Growth Factor Signaling: Identification of Src Family Kinase-Mediated Phosphorylation Events

Activation of signal transduction by the receptor tyrosine kinase, fibroblast growth factor receptor (FGFR), results in a cascade of protein−protein interactions that rely on the occurrence of specific tyrosine phosphorylation events. One such protein recruited to the activated receptor complex is the nonreceptor tyrosine kinase, Src, which is involved in both initiation and termination of further signaling events. To gain a further understanding of the tyrosine phosphorylation events that occur during FGF signaling, with a specific focus on those that are dependent on Src family kinase (SFK) activity, we have applied SILAC combined with chemical inhibition of SFK activity to search for phosphorylation events that are dependent on SFK activity in FGF stimulated cells. In addition, we used a more targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and to identify SFK-dependent Dok1 binding partners. From these analyses we identify 80 SFK-dependent phosphorylation events on 40 proteins. We further identify 18 SFK-dependent Dok1 interactions and 9 SFK-dependent Dok1 phosphorylation sites, 6 of which had not previously been known to be SFK-dependent

Summary of functional annotation and location information of the conserved common modules.

<p>Summary of functional annotation and location information of the conserved common modules.</p

Differential Phosphoproteomics of Fibroblast Growth Factor Signaling: Identification of Src Family Kinase-Mediated Phosphorylation Events

Activation of signal transduction by the receptor tyrosine kinase, fibroblast growth factor receptor (FGFR), results in a cascade of protein−protein interactions that rely on the occurrence of specific tyrosine phosphorylation events. One such protein recruited to the activated receptor complex is the nonreceptor tyrosine kinase, Src, which is involved in both initiation and termination of further signaling events. To gain a further understanding of the tyrosine phosphorylation events that occur during FGF signaling, with a specific focus on those that are dependent on Src family kinase (SFK) activity, we have applied SILAC combined with chemical inhibition of SFK activity to search for phosphorylation events that are dependent on SFK activity in FGF stimulated cells. In addition, we used a more targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and to identify SFK-dependent Dok1 binding partners. From these analyses we identify 80 SFK-dependent phosphorylation events on 40 proteins. We further identify 18 SFK-dependent Dok1 interactions and 9 SFK-dependent Dok1 phosphorylation sites, 6 of which had not previously been known to be SFK-dependent

Differential Phosphoproteomics of Fibroblast Growth Factor Signaling: Identification of Src Family Kinase-Mediated Phosphorylation Events

Activation of signal transduction by the receptor tyrosine kinase, fibroblast growth factor receptor (FGFR), results in a cascade of protein−protein interactions that rely on the occurrence of specific tyrosine phosphorylation events. One such protein recruited to the activated receptor complex is the nonreceptor tyrosine kinase, Src, which is involved in both initiation and termination of further signaling events. To gain a further understanding of the tyrosine phosphorylation events that occur during FGF signaling, with a specific focus on those that are dependent on Src family kinase (SFK) activity, we have applied SILAC combined with chemical inhibition of SFK activity to search for phosphorylation events that are dependent on SFK activity in FGF stimulated cells. In addition, we used a more targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and to identify SFK-dependent Dok1 binding partners. From these analyses we identify 80 SFK-dependent phosphorylation events on 40 proteins. We further identify 18 SFK-dependent Dok1 interactions and 9 SFK-dependent Dok1 phosphorylation sites, 6 of which had not previously been known to be SFK-dependent

FAIMS and Phosphoproteomics of Fibroblast Growth Factor Signaling: Enhanced Identification of Multiply Phosphorylated Peptides

We have applied liquid chromatography high-field asymmetric waveform ion mobility spectrometry tandem mass spectrometry (LC–FAIMS–MS/MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) to the investigation of site-specific phosphorylation in fibroblast growth factor (FGF) signaling. We have combined a SILAC approach with chemical inhibition by SU5402 (an FGF receptor tyrosine kinase inhibitor) and dasatinib (a Src family kinase inhibitor). The results show that incorporation of FAIMS within the workflow results in (a) an increase in the relative proportion of phosphothreonine and phosphotyrosine sites identified, (b) an increase in phosphopeptide identifications from precursors with charge states ≥ +3 (with an associated increase in peptide length), and (c) an increase in the identification of multiply phosphorylated peptides. Approximately 20% of the phosphorylation sites identified via the FAIMS workflow had not been reported previously, and over 80% of those were from multiply phosphorylated peptides. Moreover, FAIMS provided access to a distinct set of phosphorylation sites regulated in response to SU5402 and dasatinib. The enhanced identification of multiply phosphorylated peptides was particularly striking in the case of sites regulated by SU5402. In addition to providing a compelling example of the complementarity of FAIMS in phosphoproteomics, the results provide a valuable resource of phosphorylation sites for further investigation of FGF signaling and trafficking

Correlation of scores with B-scores.

<p>All modules with size larger than 2 and B-score are included. A few modules whose B-score is 0 (indicating scores exceeding the lower limit of detection in the B-score algorithm) were excluded. Fitted lines of versus are shown. The fitted Pearson's correlation values are 0.57 (grade II glioma, left panel) and 0.65 (GBM, right panel) respectively, with both correlation values smaller than 0.0001 in Pearson's correlation tests.</p

Differential Phosphoproteomics of Fibroblast Growth Factor Signaling: Identification of Src Family Kinase-Mediated Phosphorylation Events

Activation of signal transduction by the receptor tyrosine kinase, fibroblast growth factor receptor (FGFR), results in a cascade of protein−protein interactions that rely on the occurrence of specific tyrosine phosphorylation events. One such protein recruited to the activated receptor complex is the nonreceptor tyrosine kinase, Src, which is involved in both initiation and termination of further signaling events. To gain a further understanding of the tyrosine phosphorylation events that occur during FGF signaling, with a specific focus on those that are dependent on Src family kinase (SFK) activity, we have applied SILAC combined with chemical inhibition of SFK activity to search for phosphorylation events that are dependent on SFK activity in FGF stimulated cells. In addition, we used a more targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and to identify SFK-dependent Dok1 binding partners. From these analyses we identify 80 SFK-dependent phosphorylation events on 40 proteins. We further identify 18 SFK-dependent Dok1 interactions and 9 SFK-dependent Dok1 phosphorylation sites, 6 of which had not previously been known to be SFK-dependent

General work flow for the DiME framework.

<p>General work flow for the DiME framework.</p