Phosphopeptide Isomers Research Articles

Improving Phosphoproteomics Profiling Using Data-Independent Mass Spectrometry

Mass spectrometry-based profiling of the phosphoproteome is a powerful method of identifying phosphorylation events at a systems level. Most phosphoproteomics studies have used data-dependent acquisition (DDA) mass spectrometry as their method of choice. In this Perspective, we review some recent studies benchmarking DDA and DIA methods for phosphoproteomics and discuss data analysis options for DIA phosphoproteomics. In order to evaluate the impact of data-dependent and data-independent acquisition (DIA) on identification and quantification, we analyze a previously published phosphopeptide-enriched data set consisting of 10 replicates acquired by DDA and DIA each. We find that though more unique identifications are made in DDA data, phosphopeptides are identified more consistently across replicates in DIA. We further discuss the challenges of identifying chromatographically coeluting phosphopeptide isomers and investigate the impact on reproducibility of identifying high-confidence site-localized phosphopeptides in replicates.

Evaluating Spatiotemporal Dynamics of Phosphorylation of RNA Polymerase II Carboxy-Terminal Domain by Ultraviolet Photodissociation Mass Spectrometry.

The critical role of site-specific phosphorylation in eukaryotic transcription has motivated efforts to decipher the complex phosphorylation patterns exhibited by the carboxyl-terminal domain (CTD) of RNA polymerase II. Phosphorylation remains a challenging post-translational modification to characterize by mass spectrometry owing to the labile phosphate ester linkage and low stoichiometric prevalence, two features that complicate analysis by high-throughput MS/MS methods. Identifying phosphorylation sites represents one significant hurdle in decrypting the CTD phosphorylation, a problem exaggerated by a large number of potential phosphorylation sites. An even greater obstacle is decoding the dynamic phosphorylation pattern along the length of the periodic CTD sequence. Ultraviolet photodissociation (UVPD) is a high-energy ion activation method that provides ample backbone cleavages of peptides while preserving labile post-translational modifications that facilitate their confident localization. Herein, we report a quantitative parallel reaction monitoring (PRM) method developed to monitor spatiotemporal changes in site-specific Ser5 phosphorylation of the CTD by cyclin-dependent kinase 7 (CDK7) using UVPD for sequence identification, phosphosite localization, and differentiation of phosphopeptide isomers. We capitalize on the series of phospho-retaining fragment ions produced by UVPD to create unique transition lists that are pivotal for distinguishing the array of phosphopeptides generated from the CTD.

Accurate Quantitative Proteomic Analyses Using Metabolic Labeling and High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS).

Stable isotope labeling by amino acids in cell culture (SILAC) is routinely used to profile changes in protein and peptide abundance across different experimental paradigms. As with other quantitative proteomic approaches, the detection of peptide isotopomers can be limited by the presence of interference ions that ultimately affect the quality of quantitative measurements. Here, we evaluate high field asymmetric waveform ion mobility spectrometry (FAIMS) to improve the accuracy and dynamic range of quantitative proteomic analyses using SILAC. We compared quantitative measurements for tryptic digests of isotopically labeled protein extracts mixed in different ratios using LC-MS/MS with and without FAIMS. To further reduce sample complexity, we also examined the improvement in quantitative measurements when combining strong cation exchange (SCX) fractionation prior to LC-MS/MS analyses. Using the same amount of sample consumed, analyses performed using FAIMS provided more than 30% and 200% increase in the number of quantifiable peptides compared to LC-MS/MS performed with and without SCX fractionation, respectively. Furthermore, FAIMS reduced the occurrence of interfering isobaric ions and improved the accuracy of quantitative measurements. We leveraged the application of FAIMS in phosphoproteomic analyses to profile dynamic changes in protein phosphorylation in HEK293 cells subjected to heat shock for periods up to 20 min. In addition to the enhanced phosphoproteomic coverage, FAIMS also provided the ability to separate phosphopeptide isomers that often coelute and can be misassigned in conventional LC-MS/MS experiments.

Improved Sensitivity and Separations for Phosphopeptides using Online Liquid Chromotography Coupled with Structures for Lossless Ion Manipulations Ion Mobility-Mass Spectrometry.

Phosphoproteomics greatly augments proteomics and holds tremendous potential for insights into the modulation of biological systems for various disease states. However, numerous challenges hinder conventional methods in terms of measurement sensitivity, throughput, quantification, and capabilities for confident phosphopeptide and phosphosite identification. In this work, we report the first example of integratingstructures for lossless ion manipulations ion mobility-mass spectrometry (SLIM IM-MS) with online reversed-phase liquid chromatography (LC) to evaluate its potential for addressing the aforementioned challenges. A mixture of 51 heavy-labeled phosphopeptides was analyzed with a SLIM IM module having integrated ion accumulation and long-path separation regions. The SLIM IM-MS provided limits of detection as low as 50-100 pM (50-100 amol/μL) for several phosphopeptides, with the potential for significant further improvements. In addition, conventionally problematic phosphopeptide isomers could be resolved following an 18 m SLIM IM separation. The 2-D LC-IM peak capacity was estimated as ∼9000 for a 90 min LC separation coupled to an 18 m SLIM IM separation, considerably higher than LC alone and providing a basis for both improved identification and quantification, with additional gains projected with the future use of longer path SLIM IM separations. Thus, LC-SLIM IM-MS offers great potential for improving the sensitivity, separation, and throughput of phosphoproteomics analyses.

Exploiting charge differences for the analysis of challenging post-translational modifications by capillary electrophoresis-mass spectrometry

Reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with mass spectrometry (MS) is typically employed for mapping modifications in proteins and peptides. Here we applied a low-flow capillary electrophoresis (CE) −electrospray ionization interface coupled to Orbitrap mass spectrometers to analyze challenging modifications such as asparagine deamidation, aspartate isomerization, arginine citrullination, and phosphopeptide isomers. We achieved excellent resolution of asparagine (Asn), aspartic acid (Asp) and isoaspartic acid (iso-Asp) containing peptides using a synthetic peptide mixture. The migration order in CE enabled a clear assignment of in vitro deamidation/isomerization sites in a protein standard mixture of intermediate complexity (48 proteins) as well as the determination of the in vivo deamidation rate of histone H1.0 directly in a crude nuclear protein fraction. Besides these well-known modifications citrullination, a post-translational modification which changes the positively charged guanidinium group of arginine to the uncharged ureido group of citrulline, was investigated. Applying CE-MS for fast and sensitive analyses of various post-translational modifications of intact and enzymatically digested histone H4, we were able to detect a variety of citrullinated proteoforms. MS/MS analysis with electron transfer dissociation (ETD) fragmentation identified the presence of deiminated Arg at position 3 and 17 of histone H4. Moreover, based on CE-MS, isobaric mono-phosphorylated peptides obtained in the course of a kinase activity study were separated and individual positional isomers quantified.

Study of O-Phosphorylation Sites in Proteins Involved in Photosynthesis-Related Processes in Synechocystis sp. Strain PCC 6803: Application of the SRM Approach.

O-Phosphorylation has been shown in photosynthesis-related proteins in a cyanobacterium Synechocystis sp. strain PCC 6803 (thereafter Synechocystis 6803), suggesting that phosphorylation of S, T, and Y residues might be important in photosynthesis-related processes. Investigation of biological roles of these phosphorylation events requires confident knowledge of the phosphorylated sites and prospects for their individual assessment. We performed phosphoproteomic analysis of Synechocystis 6803 using TiO2 enrichment of the phosphopeptides, followed by LC-MS/MS, and discovered 367 phosphorylation sites in 190 proteins participating in various cellular functions. Furthermore, we focused on the large group of phosphoproteins that are involved in light harvesting, photosynthesis-driven electron flow, photoprotection, and CO2 fixation. The SRM approach was applied to verify/improve assignments of phosphorylation sites in these proteins and to investigate possibilities for analysis of phosphopeptide isomers. The SRM assays were designed for peptides comprising 45 phosphorylation sites. The assays contain peptide iRT values and Q1/Q3 transitions comprising those discriminating between phosphopeptide isoforms. The majority of investigated phosphopeptides and phosphorylated isoforms could be individually assessed with the SRM technique. The assays could be potentially used in future quantitative studies to evaluate an extent of phosphorylation in photosynthesis-related proteins in Synechocystis 6803 cells challenged with various environmental stresses.

MS1 Peptide Ion Intensity Chromatograms in MS2 (SWATH) Data Independent Acquisitions. Improving Post Acquisition Analysis of Proteomic Experiments

Quantitative analysis of discovery-based proteomic workflows now relies on high-throughput large-scale methods for identification and quantitation of proteins and post-translational modifications. Advancements in label-free quantitative techniques, using either data-dependent or data-independent mass spectrometric acquisitions, have coincided with improved instrumentation featuring greater precision, increased mass accuracy, and faster scan speeds. We recently reported on a new quantitative method called MS1 Filtering (Schilling et al. (2012) Mol. Cell. Proteomics 11, 202–214) for processing data-independent MS1 ion intensity chromatograms from peptide analytes using the Skyline software platform. In contrast, data-independent acquisitions from MS2 scans, or SWATH, can quantify all fragment ion intensities when reference spectra are available. As each SWATH acquisition cycle typically contains an MS1 scan, these two independent label-free quantitative approaches can be acquired in a single experiment. Here, we have expanded the capability of Skyline to extract both MS1 and MS2 ion intensity chromatograms from a single SWATH data-independent acquisition in an Integrated Dual Scan Analysis approach. The performance of both MS1 and MS2 data was examined in simple and complex samples using standard concentration curves. Cases of interferences in MS1 and MS2 ion intensity data were assessed, as were the differentiation and quantitation of phosphopeptide isomers in MS2 scan data. In addition, we demonstrated an approach for optimization of SWATH m/z window sizes to reduce interferences using MS1 scans as a guide. Finally, a correlation analysis was performed on both MS1 and MS2 ion intensity data obtained from SWATH acquisitions on a complex mixture using a linear model that automatically removes signals containing interferences. This work demonstrates the practical advantages of properly acquiring and processing MS1 precursor data in addition to MS2 fragment ion intensity data in a data-independent acquisition (SWATH), and provides an approach to simultaneously obtain independent measurements of relative peptide abundance from a single experiment.

Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of Streptomyces coelicolor by Targeted LC–MS/MS

The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC–MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC–MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD000095.

Site-specific separation and detection of phosphopeptide isomers with pH-mediated stacking capillary electrophoresis-electrospray ionization-tandem mass spectrometry

This study reported a pH-mediated stacking CE coupled with ESI MS/MS method to determine the phosphorylation sites of three synthetic phosphopeptides containing structural isomers. These phosphopeptides mimic the phosphopeptides (amino acid residues 12-25) derived from the trypsin-digested products of human lamin A/C protein. The LODs were determined to be 118, 132 and 1240 fmol for SGAQASS(19)TpPL(22)SPTR, SGAQASS(19)TPL(22)SpPTR, and SGAQASS(19)TpPL(22)SpPTR, respectively. The established method was employed to analyze the phosphorylation sites of the trypsin-digested products of glutathione S-transferase-lamin A/C (1-57) fusion protein that had been phosphorylated in vitro by cyclin-dependent kinase 1. The results indicated that this method is feasible to specifically determine the phosphorylation site from phosphopeptide isomers in the trypsin-digested products of a kinase-catalyzed phosphoprotein, which should benefit the investigation of protein kinase-mediated cellular signal transduction.

Occurrence and Detection of Phosphopeptide Isomers in Large-Scale Phosphoproteomics Experiments

The past decade has been marked by the emergence of selective affinity media and sensitive mass spectrometry instrumentation that facilitated large-scale phosphoproteome analyses and expanded the repertoire of protein phosphorylation. Despite these remarkable advances, the precise location of the phosphorylation site still represents a sizable challenge in view of the labile nature of the phosphoester bond and the presence of neighboring phosphorylatable residues within the same peptide. This difficulty is exacerbated by the combinatorial distribution of phosphorylated residues giving rise to different phosphopeptide isomers. These peptides have similar physicochemical properties, and their separation by LC is often problematic. Few studies have described the frequency and distribution of phosphoisomers in large-scale phosphoproteomics experiments, and no convenient informatics tools currently exist to facilitate their detection. To address this analytical challenge, we developed two algorithms to detect separated and co-eluting phosphopeptide isomers and target their subsequent identification using an inclusion list in LC-MS/MS experiments. Using these algorithms, we determined that the proportion of isomers present in phosphoproteomics studies from mouse, rat, and fly cell extracts represents 3-6% of all identified phosphopeptides. While conventional analysis can identify chromatographically separated phosphopeptides, targeted LC-MS/MS analyses using inclusion lists provided complementary identification and expanded the number of phosphopeptide isomers by at least 52%. Interestingly, these analyses revealed that the occurrence of phosphopeptides isomers can also correlate with the presence of extended phosphorylatable amino acids that can act as a "phosphorylation switch" to bind complementary domains such as those present in SR proteins and ribonucleoprotein complexes.

Extracted fragment ion mobility distributions: A new method for complex mixture analysis

A new method is presented for constructing ion mobility distributions of precursor ions based upon the extraction of drift time distributions that are monitored for selected fragment ions. The approach is demonstrated with a recently designed instrument that combines ion mobility spectrometry (IMS) with ion trap mass spectrometry (MS) and ion fragmentation, as shown in a recent publication [J. Am. Soc. Mass Spectrom. 22 (2011) 1477–1485]. Here, we illustrate the method by examining selected charge states of electrosprayed ubiquitin ions, an extract from diesel fuel, and a mixture of phosphorylated peptide isomers. For ubiquitin ions, extraction of all drift times over small mass-to-charge ( m/ z) ranges corresponding to unique fragments of a given charge state allows the determination of precursor ion mobility distributions. A second example of the utility of the approach includes the distinguishing of precursor ion mobility distributions for isobaric, basic components from commercially available diesel fuel. Extraction of data for a single fragment ion is sufficient to distinguish the precursor ion mobility distribution of cycloalkyl-pyridine derivatives from pyrindan derivatives. Finally, the method is applied for the analysis of phosphopeptide isomers (LFpTGHPESLER and LFTGHPEpSLER) in a mixture. The approach alleviates several analytical challenges that include separation and characterization of species having similar (or identical) m/ z values within complex mixtures.

Separation of multiphosphorylated peptide isomers by CZE

A separation of mono, doubly and triply phosphorylated isomers was developed with CZE with an aqueous electrolyte containing 3.9 mol/L formic acid and 30% v/v trifluoroethanol. Thus a mixture of ten phosphopeptides corresponding to the human tau sequence 226-240 was separated within 70 min. Although peptides with different phosphorylation degrees, i.e. 0-3 phosphate groups, were well separated, some of the phosphopeptide isomers containing one or two phosphate groups were only partially separated. The electrolyte system is compatible with both MALDI- and ESI-MS, allowing a direct coupling, and thus could have some interesting applications in proteomics.

Phosphopeptide isomer separation using capillary zone electrophoresis for the study of protein kinases and phosphatases.

Methods for the rapid separation of phosphopeptide isomers (peptides with the same sequence but with phosphates on different residues) were developed using capillary zone electrophoresis with ultraviolet (CZE-UV) detection. Uncoated, cationic and neutral capillaries were used with both acidic and basic peptides. These methods enabled the assay of several protein kinases (mitogen activated protein kinase, protein kinase A, GST-tyrosine kinase) and phosphatases (acid, alkaline, and protein tyrosine phosphatase) and the determination of the sites of phosphorylation and dephosphorylation. Incubations of nonphosphorylated or phosphorylated peptide with kinases or phosphatases took place directly in the instrument's autosampler and were monitored over several hours using CZE-UV.

Separation of sets of mono- and diphosphorylated peptides by reversed-phase high performance liquid chromatography

The effect of phosphorylation on the behavior of synthetic peptides in reversed-phase high performance liquid chromatography (RP-HPLC) was re-examined using sets of peptides that included four unphosphorylated, 10 monophosphorylated and 11 diphosphorylated analogs. Trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA) were compared as anionic ion-pairing reagents in water/acetonitrile gradient runs on a standard C 18 analytical column. The separation of the unphosphorylated and monophosphorylated peptides appeared to be dependent upon multiple factors: oncolumn time, and hydrophilicity of the phosphate group when TFA was used, and general hydrophobicity and partial negative charge when HFBA was used. Among the chromatograms developed in the TFA system, increased on-column times resulted in better separation. The retention times of some phosphopeptides in which the phosphoamino acids were located proximal to basic residues were reduced compared with other phosphopeptide isomers in the TFA system, indicating that local in-peptide ion bridges retain more hydrophilic character of the phosphate side-chain group at pH 1.9. In identical gradients HFBA generally separated the monophosphorylated peptides from the unphosphorylated parent analogs better than TFA. Nevertheless, most phosphopeptide isomers of the same sequences coeluted in both systems. In identical Chromatographic conditions, TFA produced sharper peaks with a considerably smoother baseline. Remarkably, diphosphorylated peptides frequently eluted as broad or very broad peaks in both systems, and their retention times were sometimes increased compared to the unphosphorylated and monophosphorylated analogs suggesting that the predictive methods for phosphopeptide retention times are not fully applicable for multiphosphorylated sequences. Our data indicate that the RP-HPLC of multiphosphorylated peptides and protein fragments need to be further investigated or expanded with unconventional mobile phases.

Serum stability of phosphopeptides

Synthetic phosphopeptides are increasingly used to mimic phosphoprotein fragments and in rational drug design. Both applications require a study of the stability of phosphopeptides in biological environments. Here we report a study of the stability of three sets of synthetic un-, mono-, and diphosphorylated peptides in diluted human serum. The synthetic peptides contained the phosphoserines or phosphothreonines in mid-chain position corresponding to immunodominant regions of abnormally hyperphosphorylated variants of the human τ protein present in the paired helical filaments of Alzheimer's disease. The peptide degradation in human serum was monitored, and the first metabolites formed were collected from reversed-phase high performance liquid chromatography (RP-HPLC). Seven of nine monophosphorylated peptides exhibited increased stability compared to the unphosphorylated parent analogs. All peptides phosphorylated at immunodominant sites displayed higher serum stability than phosphopeptide isomers of the same sequences giving evidence of the extended presence of the antigenic stimulus in the hosts. Analysis of the degradation pathway of the fastest degrading peptide family showed that the mono- and diphosphorylated peptides, like the unphosphorylated version, underwent aminopeptidase cleavage and the phosphate group remained attached to the serine and threonine side chains. These results indicate that phosphopeptides are suitable models of phosphoprotein fragments in biochemistry assays, and that phosphorylation can be a viable modification of peptide leads in drug design.

Reversed-phase high-performance liquid chromatographic separation of synthetic phosphopeptide isomers

Selectively phosphorylated synthetic peptides corresponding to the human neurofilament protein middle-sized subunit, H-Lys—Ser—Pro—Val—Pro—Lys—Ser—Pro—Val—Glu—Glu—Lys—Gly-Oh, and its analogues were separated by reversed-phase high-performance liquid chromatography of mixtures consisting of the non-phosphorylated, the diphosphorylated and the two different monophosphorylated isomers. Application of the algorithm for the expected retention times to 4–9 amino acid-long peptide fragments revealed the correct elution order of the monophosphorylated isomers. According to circular dichroism studies, this elution order is also compatible with the possibility of induced conformational orientation on the surface of the bonded phase. Chromatographic analysis of the synthetic phosphorylation reaction indicates that the reaction rates of the two structurally different monophosphorylated peptides are similar, which is in contrast to the in vivo site-directed reaction.