Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS.

Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific N-glycopeptide isoforms of AGP from control and disease plasma sample.

Proteogenomic Study beyond Chromosome 9: New Insight into Expressed Variant Proteome and Transcriptome in Human Lung Adenocarcinoma Tissues.

This is a report of a human proteome project (HPP) related to chromosome 9 (Chr 9). To reveal missing proteins and undiscovered features in proteogenomes, both LC-MS/MS analysis and next-generation RNA sequencing (RNA-seq)-based identification and characterization were conducted on five pairs of lung adenocarcinoma tumors and adjacent nontumor tissues. Before our previous Chromosome-Centric Human Proteome Project (C-HPP) special issue, there were 170 remaining missing proteins on Chr 9 (neXtProt 2013.09.26 rel.); 133 remain at present (neXtProt 2015.04.28 rel.). In the proteomics study, we found two missing protein candidates that require follow-up work and one unrevealed protein across all chromosomes. RNA-seq analysis detected RNA expression for four nonsynonymous (NS) single nucleotide polymorphisms (SNPs) (in CDH17, HIST1H1T, SAPCD2, and ZNF695) and three synonymous SNPs (in CDH17, CST1, and HNF1A) in all five tumor tissues but not in any of the adjacent normal tissues. By constructing a cancer patient sample-specific protein database based on individual RNA-seq data and by searching the proteomics data from the same sample, we identified four missense mutations in four genes (LTF, HDLBP, TF, and HBD). Two of these mutations were found in tumor samples but not in paired normal tissues. In summary, our proteogenomic study of human primary lung tumor tissues detected additional and revealed novel missense mutations and synonymous SNP signatures, some of which are specific to lung cancers. Data from mass spectrometry have been deposited in the ProteomeXchange with the identifier PXD002523.

Crystal structure of bet3 reveals a novel mechanism for Golgi localization of tethering factor TRAPP.

Transport protein particle (TRAPP) is a large multiprotein complex involved in endoplasmic reticulum-to-Golgi and intra-Golgi traffic. TRAPP specifically and persistently resides on Golgi membranes. Neither the mechanism of the subcellular localization nor the function of any of the individual TRAPP components is known. Here, the crystal structure of mouse Bet3p (bet3), a conserved TRAPP component, reveals a dimeric structure with hydrophobic channels. The channel entrances are located on a putative membrane-interacting surface that is distinctively flat, wide and decorated with positively charged residues. Charge-inversion mutations on the flat surface of the highly conserved yeast Bet3p led to conditional lethality, incorrect localization and membrane trafficking defects. A channel-blocking mutation led to similar defects. These data delineate a molecular mechanism of Golgi-specific targeting and anchoring of Bet3p involving the charged surface and insertion of a Golgi-specific hydrophobic moiety into the channels. This essential subunit could then direct other TRAPP components to the Golgi.

A splice variant of the C(2)H(2)-type zinc finger protein, ZNF268s, regulates NF-kappaB activation by TNF-alpha.

IkappaB kinase (IKK), the pivotal kinase in signal-dependent activation of nuclear factor-kappaB (NF-kappaB), is composed of multiple protein components, including IKK alpha/beta/gamma core subunits. To investigate the regulation of the IKK complex, we immunoaffinity purified the IKK complex, and by MALDI-TOF mass spectrometry identified a splice variant of zinc finger protein 268 (ZNF268) as a novel IKK-interacting protein. Both the full-length and the spliced form of the ZNF268 protein were detected in a variety of mammalian tissues and cell lines. The genes were cloned and expressed by in vitro transcription/translation. Several deletion derivatives, such as KRAB domain (KRAB) on its own, the KRAB/spacer/4-zinc fingers (zF4), and the spacer/ 4-zinc fingers (zS4), were ectopically expressed in mammalian cells and exhibited had different subcellular locations. The KRAB-containing mutants were restricted to the nucleus, while zS4 was localized in the cytosol. TNF-alpha-induced NF-kappaB activation was examined using these mutants and only zS4 was found to stimulate activation. Collectively, the results indicate that a spliced form of ZNF268 lacking the KRAB domain is located in the cytosol, where it seems to play a role in TNF-alpha-induced NF-kappaB activation by interacting with the IKK complex.

MODi: a powerful and convenient web server for identifying multiple post-translational peptide modifications from tandem mass spectra.

MOD(i) (http://modi.uos.ac.kr/modi/) is a powerful and convenient web service that facilitates the interpretation of tandem mass spectra for identifying post-translational modifications (PTMs) in a peptide. It is powerful in that it can interpret a tandem mass spectrum even when hundreds of modification types are considered and the number of potential PTMs in a peptide is large, in contrast to most of the methods currently available for spectra interpretation that limit the number of PTM sites and types being used for PTM analysis. For example, using MOD(i), one can consider for analysis both the entire PTM list published on the unimod webpage (http://www.unimod.org) and user-defined PTMs simultaneously, and one can also identify multiple PTM sites in a spectrum. MOD(i) is convenient in that it can take various input file formats such as .mzXML, .dta, .pkl and .mgf files, and it is equipped with a graphical tool called MassPective developed to display MOD(i)'s output in a user-friendly manner and helps users understand MOD(i)'s output quickly. In addition, one can perform manual de novo sequencing using MassPective.

Post-translational modifications and their biological functions: proteomic analysis and systematic approaches.

Recently produced information on post-translational modifications makes it possible to interpret their biological regulation with new insights. Various protein modifications finely tune the cellular functions of each protein. Understanding the relationship between post-translational modifications and functional changes ("post-translatomics") is another enormous project, not unlike the human genome project. Proteomics, combined with separation technology and mass spectrometry, makes it possible to dissect and characterize the individual parts of post-translational modifications and provide a systemic analysis. Systemic analysis of post-translational modifications in various signaling pathways has been applied to illustrate the kinetics of modifications. Availability will advance new technologies that improve sensitivity and peptide coverage. The progress of "post-translatomics", novel analytical technologies that are rapidly emerging, offer a great potential for determining the details of the modification sites.

N-terminal truncated UCH-L1 prevents Parkinson's disease associated damage.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been proposed as one of the Parkinson's disease (PD) related genes, but the possible molecular connection between UCH-L1 and PD is not well understood. In this study, we discovered an N-terminal 11 amino acid truncated variant UCH-L1 that we called NT-UCH-L1, in mouse brain tissue as well as in NCI-H157 lung cancer and SH-SY5Y neuroblastoma cell lines. In vivo experiments and hydrogen-deuterium exchange (HDX) with tandem mass spectrometry (MS) studies showed that NT-UCH-L1 is readily aggregated and degraded, and has more flexible structure than UCH-L1. Post-translational modifications including monoubiquitination and disulfide crosslinking regulate the stability and cellular localization of NT-UCH-L1, as confirmed by mutational and proteomic studies. Stable expression of NT-UCH-L1 decreases cellular ROS levels and protects cells from H2O2, rotenone and CCCP-induced cell death. NT-UCH-L1-expressing transgenic mice are less susceptible to degeneration of nigrostriatal dopaminergic neurons seen in the MPTP mouse model of PD, in comparison to control animals. These results suggest that NT-UCH-L1 may have the potential to prevent neural damage in diseases like PD.

Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: application to glyceraldehyde-3-phosphate dehydrogenase.

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.

Proteomic analysis of tyrosine phosphorylations in vascular endothelial growth factor- and reactive oxygen species-mediated signaling pathway.

Vascular endothelial growth factor (VEGF) mediates angiogenic signaling by activating tyrosine kinase receptors. Endothelial cells treated with VEGF are known to increase reactive oxygen species (ROS) production and activate the MAPK pathway. To identify the target proteins of the VEGF receptor, we treated human umbilical vein endothelial cells (HUVECs) with VEGF or H2O2, and identified and semiquantified tyrosine-phosphorylated proteins, combining 2D-gel electrophoresis, Western analysis using antibody against phospho-tyrosine, and mass spectrometry. We detected 95 proteins that were differentially phosphorylated; some were specifically phosphorylated by VEGF but not by H2O2. 2D-gel electrophoresis revealed that heterogeneous populations of the same protein responded differently to H2O2 and VEGF. Bioinformatic studies examining the nature of the differential phosphorylation in various subpopulations of proteins should provide new insights into VEGF- and H2O2-induced signaling pathways.

Systemic analysis of tyrosine phosphorylated proteins in angiopoietin-1 induced signaling pathway of endothelial cells.

Angiogenesis is an essential process in physiological and pathological processes and is well-regulated to maintain the cellular homeostasis by balancing the endothelial cells in proliferation and apoptosis. Angiopoietin-1 (Ang1) regulates angiogenesis as a ligand of Tie 2 receptor tyrosine kinase. However, the regulation pathways are not well-understood. To date, only a few of the signaling molecules involved in the Tie 2 receptor tyrosine kinase-mediated angiogenesis have been identified. In this study, we systematically identified tyrosine-phosphorylated proteins in Ang1-induced signaling cascade in human umbilical vein endothelial cells (HUVECs), employing proteomic analyses combining two-dimensional gel electrophoresis, Western analysis using phosphotyrosine antibody and mass spectrometry (MALDI-TOF MS and nanoLC-ESI-q-TOF tandem MS). We report here the identification, semiquantitative analysis, and kinetic changes of tyrosine-phosphorylated proteins in response to Ang1 in HUVECs and identified 66 proteins among 69 protein spots showing significant changes. Of these, p54nrb was validated as a molecule involved in cell migration. These results suggest that Ang1 induces stabilization of neo-vessel network by regulating the phosphorylations of metabolic and structural proteins.