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Quantitative phosphoproteomics has transformed investigations of cell signaling, but it remains challenging to scale the technology for high-throughput analyses. Here we report a rapid and reproducible approach to analyze hundreds of phosphoproteomes using data-independent acquisition (DIA) with an accurate site localization score incorporated into Spectronaut. DIA-based phosphoproteomics achieves an order of magnitude broader dynamic range, higher reproducibility of identification, and improved sensitivity and accuracy of quantification compared to state-of-the-art data-dependent acquisition (DDA)-based phosphoproteomics. Notably, direct DIA without the need of spectral libraries performs close to analyses using project-specific libraries, quantifying > 20,000 phosphopeptides in 15 min single-shot LC-MS analysis per condition. Adaptation of a 3D multiple regression model-based algorithm enables global determination of phosphorylation site stoichiometry in DIA. Scalability of the DIA approach is demonstrated by systematically analyzing the effects of thirty kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that specific protein kinases mediate EGF-dependent phospho-regulation. Localizing phosphorylation sites by data-independent acquisition (DIA)-based proteomics is still challenging. Here, the authors develop algorithms for phosphosite localization and stoichiometry determination, and incorporate them into single-shot DIA-phosphoproteomics workflows.

Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible quantification remains challenging, especially for post-translational modifications such as phosphorylation. Here, we compare the most popular quantification techniques for global phosphoproteomics: label-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS 2 - and MS 3 -measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide ratios, we find LFQ and SILAC to be the most accurate techniques. MS 2 -based TMT yields the highest precision but lowest accuracy due to ratio compression, which MS 3 -based TMT can partly rescue. However, MS 2 -based TMT outperforms MS 3 -based TMT when analyzing phosphoproteome changes in the DNA damage response, since its higher precision and larger identification numbers allow detection of a greater number of significantly regulated phosphopeptides. Finally, we utilize the TMT multiplexing capabilities to develop an algorithm for determining phosphorylation site stoichiometry, showing that such applications benefit from the high accuracy of MS 3 -based TMT. Quantitative phosphoproteomics has become a standard method in molecular and cell biology. Here, the authors compare performance and parameters of phosphoproteome quantification by LFQ, SILAC, and MS 2 -/MS 3 -based TMT and introduce a TMT-adapted algorithm for calculating phosphorylation site stoichiometry.

Phosphoproteomics integrating data-independent acquisition (DIA) enables deep phosphoproteome profiling with improved quantification reproducibility and accuracy compared to data-dependent acquisition (DDA)-based phosphoproteomics. DIA data mining heavily relies on a spectral library that in most cases is built on DDA analysis of the same sample. Construction of this project-specific DDA library impairs the analytical throughput, limits the proteome coverage, and increases the sample size for DIA phosphoproteomics. Herein we introduce a deep neural network, DeepPhospho, which conceptually differs from previous deep learning models to achieve accurate predictions of LC-MS/MS data for phosphopeptides. By leveraging in silico libraries generated by DeepPhospho, we establish a DIA workflow for phosphoproteome profiling which involves DIA data acquisition and data mining with DeepPhospho predicted libraries, thus circumventing the need of DDA library construction. Our DeepPhospho-empowered workflow substantially expands the phosphoproteome coverage while maintaining high quantification performance, which leads to the discovery of more signaling pathways and regulated kinases in an EGF signaling study than the DDA library-based approach. DeepPhospho is provided as a web server as well as an offline app to facilitate user access to model training, predictions and library generation. The coverage and throughput of data-independent acquisition (DIA)-based phosphoproteomics is limited by its dependence on experimental spectral libraries. Here the authors develop a DIA workflow based on in silico spectral libraries generated by a novel deep neural network to expand phosphoproteome coverage.

Protein N-phosphorylation plays a critical role in central metabolism and two/multicomponent signaling of prokaryotes. However, the current enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides due to the intrinsic lability of P-N bond under acidic conditions. Therefore, the effective N-phosphoproteome analysis remains challenging. Herein, bis(zinc(II)-dipicolylamine)-functionalized sub-2 μm core-shell silica microspheres (SiO 2 @DpaZn) are tailored for rapid and effective N-phosphopeptides enrichment. Due to the coordination of phosphate groups to Zn(II), N-phosphopeptides can be effectively captured under neutral conditions. Moreover, the method is successfully applied to an E.coli and HeLa N-phosphoproteome study. These results further broaden the range of methods for the discovery of N-phosphoproteins with significant biological functions. N-phosphorylation plays a critical role in central metabolism and signaling processes, however, enrichment methods for N-phosphopeptides are limited by the P-N bond lability. Here, the authors report the synthesis and use of silica microspheres functionalized with bis(zinc(II)-dipicolylamine) in N-phosphopeptides effective enrichment.

A novel cerium phthalocyanine-based covalent organic framework (CePc-COF)-magnetic core–shell composite was fabricated by grafting the CePc-COF layer on the surface of Fe 3 O 4 . The obtained core–shell composite particle (Fe 3 O 4 @CePc-COF) had strong magnetic responsiveness (29.6 emu g −1 ) and good hydrophilicity (5.0°). The affinity material provided abundant metal ion sites for specific enrichment of phosphopeptides. Fe 3 O 4 @CePc-COF had good performance in terms of high selectivity (1: 1: 5000), sensitivity (0.1 fmol), recovery (92.91%), and reusability (10 cycles). The enrichment feasibility of Fe 3 O 4 @CePc-COF was first investigated in standard peptides. Furthermore, Fe 3 O 4 @CePc-COF can efficiently identify phosphopeptides from extremely complex samples. The work can provide a novel idea for the fabrication of metallophthalocyanine based-COF materials for phosproteome detection in biological samples. Graphical Abstract

Determining the phosphorylation of low-abundance peptides in small amount of clinical samples is challenging. In this approach, in-house construction of IMAC columns plus esterification of the peptides are the key features that improve sensitivity. Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry–compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called 'CAD Neutral Loss Finder' that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d.

CK2 denotes a ubiquitous and pleiotropic protein kinase whose holoenzyme is composed of two catalytic (α and/or α′) and two regulatory β subunits. The CK2 consensus sequence, S/T-x-x-D/E/pS/pT is present in numerous phosphosites, but it is not clear how many of these are really generated by CK2. To gain information about this issue, advantage has been taken of C2C12 cells entirely deprived of both CK2 catalytic subunits by the CRISPR/Cas9 methodology. A comparative SILAC phosphoproteomics analysis reveals that, although about 30% of the quantified phosphosites do conform to the CK2 consensus, only one-third of these are substantially reduced in the CK2α/α′ (−/−) cells, consistent with their generation by CK2. A parallel study with C2C12 cells deprived of the regulatory β subunit discloses a role of this subunit in determining CK2 targeting. We also find that phosphosites notoriously generated by CK2 are not fully abrogated in CK2α/α′ (−/−) cells, while some phosphosites unrelated to CK2 are significantly altered. Collectively taken our data allow to conclude that the phosphoproteome generated by CK2 is not as ample and rigidly pre-determined as it was believed before. They also show that the lack of CK2 promotes phosphoproteomics perturbations attributable to kinases other than CK2.

Magnetic microspheres (Fe 3 O 4 ) were coated with polydopamine (PDA) and loaded with the metal ions Ti(IV) and Nb(V) to give a material of type Fe 3 O 4 @PDA-Ti/Nb. It is shown to be useful for affinity chromatography and for enrichment of phosphopeptides from both standard protein solutions and real samples. For comparison, such microspheres loaded with single metal ions only (Fe 3 O 4 @PDA-Ti and Fe 3 O 4 @PDA-Nb) and their physical mixtures were also investigated under identical conditions. The binary metal ion-loaded magnetic microspheres display better enrichment efficiency than the single metal ion-loaded microspheres and their physical mixture. Both multiphosphopeptides and monophosphopeptides can be extracted. The Fe 3 O 4 @PDA-Ti/Nb microspheres exhibit ultra-high sensitivity (the lowest detection amount being 2 fmol) and selectivity at a low mass ratio such as in case of β-casein/BSA (1:1000). Graphical abstract Magnetic microspheres (Fe 3 O 4 ) were coated with polydopamine (PDA) and loaded with the metal ions Ti(IV) and Nb(V) to give a material of type Fe 3 O 4 @PDA-Ti/Nb. Results showed its great potential as an affinity probe in phosphoproteome research due to rapid magnetic separation of phosphopeptides, ultrahigh sensitivity and selectivity, and remarkable reusability.

In this study, a general approach to prepare metal-organic framework (MOF)-based heterogeneous catalysts is proposed. The Ce -modified MOFs were obtained by the co-precipitation of catalytic active Ce ions and catalytic inactive MOFs (ZIF-67, MOF-5, and ZIF-8). The Ce -modified MOFs retained the phosphatase-like activity of Ce ions and were used for the fluorescent detection of phosphorylated amino acids by using o-phospho-L-tyrosine (p-Tyr) as a model target. Using Ce -modified ZIF-67 particles as the heterogeneous catalysts, the linear range for p-Tyr detection is 1-10 µM with a detection limit of 0.26 µM. Compared with homogeneous Ce ion catalysts, a significant improvement in the sensitivity was achieved by using Ce -modified ZIF-67 particles as heterogeneous catalysts (from 11.21 to 0.26 µM). As the proposed method holds great promise in the fluorescent detection of phosphorylated amino acids, the Ce -modified ZIF-67-based catalytic system was further used for the discrimination of normal peptides and phosphorylated peptides with excellent resolution.

Free radical–initiated peptide sequencing mass spectrometry (FRIPS MS) was employed to analyze a number of representative singly or doubly protonated phosphopeptides (phosphoserine and phosphotyrosine peptides) in positive ion mode. In contrast to collision-activated dissociation (CAD) results, a loss of a phosphate group occurred to a limited degree for both phosphoserine and phosphotyrosine peptides, and thus, localization of a phosphorylated site was readily achieved. Considering that FRIPS MS supplies a substantial amount of collisional energy to peptides, this result was quite unexpected because a labile phosphate group was conserved. Analysis of the resulting peptide fragments revealed the extensive production of a-, c-, x-, and z-type fragments (with some minor b- and y-type fragments), suggesting that radical-driven peptide fragmentation was the primary mechanism involved in the FRIPS MS of phosphopeptides. Results of this study clearly indicate that FRIPS MS is a promising tool for the characterization of post-translational modifications such as phosphorylation.Graphical Abstractᅟ