Deep Phosphotyrosine Proteomics by Optimization of Phosphotyrosine Enrichment and MS/MS Parameters

Phosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography–tandem mass spectrometry (LC–MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC–MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, >30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anticancer targets

Proteome-Wide Discovery of Unknown ATP-Binding Proteins and Kinase Inhibitor Target Proteins Using an ATP Probe

ATP-binding proteins, including protein kinases, play essential roles in many biological and pathological processes and thus these proteins are attractive as drug targets. Acyl-ATP probes have been developed as efficient probes for kinase enrichment, and these probes have also been used to enrich other ATP-binding proteins. However, a robust method to identify ATP-binding proteins with systematic elimination of nonspecific binding proteins has yet to be established. Here, we describe an ATP competition assay that permitted establishment of a rigorous ATP-binding protein list with virtual elimination of nonspecific proteins. A total of 539 ATP-binding protein candidates were identified, including 178 novel candidates. In informatics analysis, ribosomal proteins were overrepresented in the list of novel candidates. We also found multiple ATP-competitive sites for several kinases, including epidermal growth factor receptor, serine/threonine-protein kinase PRP4 homologue, cyclin-dependent kinase 12, eukaryotic elongation factor 2 kinase, ribosomal protein S6 kinase alpha-1, and SRSF protein kinase 1. Using our cataloged ATP-binding protein list, a selectivity profiling method that covers the kinome and ATPome was established to identify off-target binding sites of ATP-competitive kinase inhibitors, staurosporine and crizotinib

In-depth Membrane Proteomic Study of Breast Cancer Tissues for the Generation of a Chromosome-based Protein List

The Chromosome-centric Human Proteome Project (C-HPP) aims to define all proteins encoded in each chromosome and especially to identify proteins that currently lack evidence by mass spectrometry. The C-HPP also prioritizes particular protein subsets such as membrane proteins, post-translational modifications, and low-abundance proteins. In this study, we aimed to generate deep profiling of the membrane proteins of human breast cancer tissues on a chromosome-by-chromosome basis using shotgun proteomics. We identified 7092 unique proteins using membrane fractions isolated from pooled breast cancer tissues with high confidence. A total of 3282 proteins were annotated as membrane proteins by Gene Ontology analysis, which covered 45% of the membrane proteins predicted in 20 859 protein-coding genes. Furthermore, we were able to identify 851 membrane proteins that currently lack evidence by mass spectrometry in neXtProt. Our results will contribute to the accomplishment of the primary goal of the C-HPP in identifying so-called “missing proteins” and generating a whole protein catalog for each chromosome

Identification of Missing Proteins in the neXtProt Database and Unregistered Phosphopeptides in the PhosphoSitePlus Database As Part of the Chromosome-Centric Human Proteome Project

The Chromosome-Centric Human Proteome Project (C-HPP) is an international effort for creating an annotated proteomic catalog for each chromosome. The first step of the C-HPP project is to find evidence of expression of all proteins encoded on each chromosome. C-HPP also prioritizes particular protein subsets, such as those with post-translational modifications (PTMs) and those found in low abundance. As participants in C-HPP, we integrated proteomic and phosphoproteomic analysis results from chromosome-independent biomarker discovery research to create a chromosome-based list of proteins and phosphorylation sites. Data were integrated from five independent colorectal cancer (CRC) samples (three types of clinical tissue and two types of cell lines) and lead to the identification of 11,278 proteins, including 8,305 phosphoproteins and 28,205 phosphorylation sites; all of these were categorized on a chromosome-by-chromosome basis. In total, 3,033 “missing proteins”, i.e., proteins that currently lack evidence by mass spectrometry, in the neXtProt database and 12,852 unknown phosphorylation sites not registered in the PhosphoSitePlus database were identified. Our in-depth phosphoproteomic study represents a significant contribution to C-HPP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD00008

Absolute Quantitation of Low Abundance Plasma APL1β peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment

Selected/multiple reaction monitoring (SRM/MRM) has been widely used for the quantification of specific proteins/peptides, although it is still challenging to quantitate low abundant proteins/peptides in complex samples such as plasma/serum. To overcome this problem, enrichment of target proteins/peptides is needed, such as immunoprecipitation; however, this is labor-intense and generation of antibodies is highly expensive. In this study, we attempted to quantify plasma low abundant APLP1-derived Aβ-like peptides (APL1β), a surrogate marker for Alzheimer’s disease, by SRM/MRM using stable isotope-labeled reference peptides without immunoaffinity enrichment. A combination of Cibacron Blue dye mediated albumin removal and acetonitrile extraction followed by C₁₈-strong cation exchange multi-StageTip purification was used to deplete plasma proteins and unnecessary peptides. Optimal and validated precursor ions to fragment ion transitions of APL1β were developed on a triple quadruple mass spectrometer, and the nanoliquid chromatography gradient for peptide separation was optimized to minimize the biological interference of plasma. Using the stable isotope-labeled (SI) peptide as an internal control, absolute concentrations of plasma APL1β peptide could be quantified as several hundred amol/mL. To our knowledge, this is the lowest detection level of endogenous plasma peptide quantified by SRM/MRM

Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient

Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques

Protein profiling between metastasized tumors of control and SB-431542-treated groups.

<p>(A) A PCA plot based on the peptide profile in control and SB-431542-treated tumors using SMCA13 for the multivariate analysis and (B) corresponding loading plot. (C) OPLS-DA plot discriminates proteins from those two groups. Black dots represent the control group (n = 4) and white dots represent the SB-431542-treated group (n = 4). (D) S-plot shows the significance of protein variations between control and SB-431542-treated groups. Each black dot represents individual proteins. Black dots in the positive directions indicate the decreased proteins in SB-431542-treated tumors. The increased proteins in SB-431542-treated tumors are presented as dots in the negative direction. Vimentin (Vim), Hsp90ab1 and Eno1 were double-line-circled as the top proteins of the highest confidence and greatest contribution separation between the untreated and SB-431542-treated mice. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126483#pone.0126483.s005" target="_blank">S1 Table</a>). A magnified picture of the significantly reduced proteins in SB-431542-treated tumors is shown (p(corr) > 0.80).</p

Validation of protein expression using western blot and immunohistochemistry.

<p>(A) Metastases lysates were prepared from three mice per group and evaluated by western blot with anti-vimentin, anti-Hsp90a/b, and anti-Eno1 antibodies. β-actin was used as internal normalization. (B) Representative image of positive vimentin staining is shown exclusively in the metastases lesion and not in the surrounding lung tissue. White dotted lines represent a boundary of tumor and surrounding normal lung tissue. M, metastases; NT, normal lung tissue; PV, pulmonary vein. (C) Randomly selected high power fields were immunostained with vimentin and were quantitated using Zeiss software with the % area occupied by metastasized nodules in 4T1 metastasized tumors. A significant reduction in the number of vimentin positive cells was seen in the SB-431542-treated tumors. Scale bars represent 100 μm; hpf, high power fields.</p

Orthotopic metastasis model demonstrates that a specific inhibitor of TGF-β receptor kinase, SB-431542, decreases lung metastasis but does not significantly alter growth of the primary tumor.

<p>(A) Scheme for the experimental approach using 4T1 metastasis model. 4T1 tumor cells (1×10⁴ cells) were transplanted into the mammary fat pad of Balb/c mice. Mice bearing 4T1 mammary tumors were treated three times weekly with SB-431542 (10 mg/kg body weight) or vehicle (20% DMSO/80% corn oil). At day 10 post-injection of 4T1 cells, primary tumors were surgically excised, and the mice were kept alive to allow the tumor to metastasize to the lung. (B) Graph showing relative primary tumor growth of 4T1 cells over time. Data are presented as mean ± SEM. (ns = not significant, control, n = 14; SB-431542, n = 15, unpaired <i>t</i>-test). (C) The number of gross metastasis (control, n = 14; SB-431542-treated, n = 15) was counted. The administration of SB-431542 markedly reduced both the number and the size of the metastasized tumor compared to the control group. Lungs were collected after 40 days and the lung surface was examined for the metastasis. The number of visible lung metastasis was counted. ** p<0.001.; Mann Whitney <i>U</i>-test. Data are presented as the median ± SEM. (D) Representative gross lung images from control and SB-431542-treated groups are shown from control (top) and SB-431542-treated (bottom) animals, with metastases visible at the lung surface marked by bold black arrows. (E) Representative histological view of lung metastases treated with vehicle, DMSO (left) and SB-431542 (right). H&E staining; black dotted lines demarcate tumor parenchyma (*) from the normal lung tissue.</p

EIF signaling was the most enriched canonical IPA pathway of the down-regulated proteins in SB-431542-treated groups.

<p>(A) Pathway analysis conducted using IPA (<a href="http://www.ingenuitypathway.com" target="_blank">www.ingenuitypathway.com</a>) showed a ranking of the most enriched pathways from the down-regulated proteins in SB-431542-treated groups. (B) Whole cell lysates from each group were evaluated by western blot with the EIF family of proteins: anti-eIF4G1, anti-eIF4E, and anti-Eef2 antibodies. (n = 3/group).β -actin was used as a loading control. (C) Sections were assessed by H&E staining (top panel), and immunohistochemistry was performed using anti-eIF4G1 (middle panel) and anti-eIF4E (bottom panel) antibodies. Both eIF4G1 and eIF4E immunostaining in 4T1 lung metastasis showed the heterogeneity of staining patterns seen among individual metastases. 100× magnification. Tumor sections are boxed with a black dotted line, M, metastases; NT, normal lung tissue. (D) eIF4G1- and eIF4E-positive areas were semiquantified using Image Pro Premier Software in three randomly selected high power fields from each samples. Data are represented as box plots (n = 3/group), ** p<0.001 using an unpaired <i>t</i>-test.</p