Konstantinos Petritis

The Drosophila melanogaster sperm proteome-II (DmSP-II)

Advances in mass spectrometry technology, high-throughput proteomics and genome annotations have resulted in significant increases in our molecular understanding of sperm composition. Using improved separation and detection methods and an updated genome annotation, a re-analysis of the Drosophila melanogaster sperm proteome (DmSP) has resulted in the identification of 956 sperm proteins. Comparative analysis with our previous proteomic dataset revealed 766 new proteins and an updated sperm proteome containing a total of 1108 proteins, termed the DmSP-II. This expanded dataset includes additional proteins with predicted sperm functions and confirms previous findings concerning the genomic organization of sperm loci. Bioinformatic and protein network analyses demonstrated high quality and reproducibility of proteome coverage across three replicate mass spectrometry runs. The use of whole-cell proteomics in conjunction with characterized phenotypes, functional annotations and pathway information has advanced our systems level understanding of sperm proteome functional networks.

The Rhesus Macaque (Macaca mulatta) Sperm Proteome

Mass spectrometry based proteomics has facilitated sperm composition studies in several mammalian species but no studies have been undertaken in non-human primate species. Here we report the analysis of the 1247 proteins that comprise the Rhesus macaque (Macaca mulatta) sperm proteome (termed the MacSP). Comparative analysis with previously characterized mouse and human sperm proteomes reveals substantial levels of orthology (47% and 40% respectively) and widespread overlap of functional categories based on Gene Ontology analyses. Approximately 10% of macaque sperm genes (113/1247) are significantly under-expressed in the testis as compared with other tissues, which may reflect proteins specifically acquired during epididymal maturation. Phylogenetic and genomic analyses of three MacSP ADAMs (A-Disintegrin and Metalloprotease proteins), ADAM18-, 20- and 21-like, provides empirical support for sperm genes functioning in non-human primate taxa which have been subsequently lost in the lineages leading to humans. The MacSP contains proteasome proteins of the 20S core subunit, the 19S proteasome activator complex and an alternate proteasome activator PA200, raising the possibility that proteasome activity is present in mature sperm. Robust empirical characterization of the Rhesus sperm proteome should greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility.

MRCQuant- an accurate LC-MS relative isotopic quantification algorithm on TOF instruments

BackgroundRelative isotope abundance quantification, which can be used for peptide identification and differential peptide quantification, plays an important role in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. However, several major issues exist in the relative isotopic quantification of peptides on time-of-flight (TOF) instruments: LC peak boundary detection, thermal noise suppression, interference removal and mass drift correction. We propose to use the Maximum Ratio Combining (MRC) method to extract MS signal templates for interference detection/removal and LC peak boundary detection. In our method, MRCQuant, MS templates are extracted directly from experimental values, and the mass drift in each LC-MS run is automatically captured and compensated. We compared the quantification accuracy of MRCQuant to that of another representative LC-MS quantification algorithm (msInspect) using datasets downloaded from a public data repository.ResultsMRCQuant showed significant improvement in the number of accurately quantified peptides.ConclusionsMRCQuant effectively addresses major issues in the relative quantification of LC-MS-based proteomics data, and it provides improved performance in the quantification of low abundance peptides.

Differential effects of AKT1(p.E17K) expression on human mammary luminal epithelial and myoepithelial cells

Recently, we identified a somatic mutation in AKT1, which results in a glutamic acid to lysine substitution (p.Glu17Lys or E17K). E17K mutations appear almost exclusively in breast cancers of luminal origin. Cellular models involving cell lines such as human mammary epithelial and MCF10 are model systems that upon transformation lead to rare forms of human breast cancer. Hence, we studied the effects of E17K using a clinically pertinent luminal cell line model while providing evidence to explain why E17K mutations do not occur in the mammary myoepithelium. Thus the purpose of our study was to perform a functional and differential proteomics study to assess the role of AKT1(E17K) in the development of breast cancer. We used a set of genetically matched nontumorigenic and tumorigenic mammary luminal and myoepithelial cells. We demonstrated that in myoepithelial cells, expression of E17K inhibited growth, migration, and protein synthesis compared with wild‐type AKT1. In luminal cells, E17K enhanced cell survival and migration, possibly offering a selective advantage in this type of cell. However, antineoplastic effects of E17K in luminal cells, such as inhibition of growth and protein synthesis, may ultimately be associated with favorable prognosis. Our study illustrates the importance of cellular context in determining phenotypic effects of putative oncogenic mutations. Hum Mutat 33:1216–1227, 2012.

Regulation of synaptojanin 2 5′-phosphatase activity by Src

Synaptojanin 2 (SYNJ2) is a phosphatidylinositol (PI) phosphatase that controls two distinct functions, clathrin-mediated endocytosis and tumor cell invadopodia formation and invasion. Here, we identify a number of novel SYNJ2 binding partners, several of which have previously been shown to be necessary for invadopodia formation or clathrin-mediated endocytosis. We focus on Src family kinases. We found that Src phosphorylates SYNJ2 on Tyr490, thereby stimulating SYNJ2 5′-phosphatase activity in vitro. We also provide evidence that Src-mediated phosphorylation of SYNJ2 contributes to invadopodia formation.

Isolation and characterization of muscle fatigue substance with anti-tumor activities.

Research during the 1950s indicated that exercise played a role in the reduction of tumor growth. In the 1960s our studies confirmed that tumor-bearing rats, exercised to fatigue, demonstrated tumor inhibition. Our further studies isolated an extract (Fatigue Substance, or F-Substance) from rectus femoris muscles of rats which had been electrically stimulated to fatigue. This extract significantly inhibited growth of transplanted rat tumors. Research continued until 1978 when it became apparent the methodology at that time was not able to further identify the substances active components. Using current technology, we now report on the further isolation and characterization of F-Substance. In cell proliferation assays, extracts from electrically stimulated rat rectus femoris muscles had more significant inhibitory effect on the breast cancer cell line MCF-7 than those isolated from unstimulated muscles. To identify the molecule(s) responsible for the antitumor activity, a rat cytokine antibody array was used to profile the cytokines in the substances. Among the 29 different cytokines contained on the array, 3 showed greater than 3-fold difference between the substances isolated from the stimulated and unstimulated muscles. LIX (also known as CXCL5) is 6-fold higher in the substances isolated from stimulated muscles than those from the unstimulated muscles. TIMP-1 is 4.6 fold higher and sICAM is 3.6 fold higher in the substances from the stimulated muscles. Our results indicated that cytokines released from contracting muscles might be responsible for the antitumor effect of F-Substance.

Accurate LC Peak Boundary Detection for 16O/18O Labeled LC-MS Data

In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges. In addition, we remove peptides with erroneous mass assignment through model fitness check, which compares observed intensity patterns to theoretically constructed ones. The proposed algorithm can significantly improve the accuracy and precision of peptide ratio measurements.

Improved Quantification of Labeled LC-MS

A novel quantification method is proposed for labeled LC-MS data. Unlike the traditional extracted ion chromatogram based quantification methods, the new method considers the reliability or the quality of signals with different intensities and applies more weights to the more reliable signals in the relative quantification procedure. Testing shows that the new method has improved accuracy than current quantification software.

Abstract 1060: A functional and proteomics analysis of AKT1(E17K) in human mammary epithelial cells of luminal and myoepithelial cells of origin

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The normal breast epithelium, from which most breast cancers arise, is composed of an inner layer of luminal cells and an outer layer of myoepithelial (basal) cells. A cell culture method recently described allows direct comparison of two genetically matched cells derived from normal myoepithelial and luminal epithelial cell types termed HME and BPE cells respectively (Ince et al, Cancer Cell 2007). Transformation of these cells (subsequently termed HMLER and BPLER) formed tumor xenografts exhibiting major differences in histopathology, tumorigenicity, and metastatic behavior. The non-receptor serine/threonine kinase AKT/PKB is one of the most commonly deregulated pathways in human breast cancer. In this regard we performed functional and proteomics assays to compare the role of wild-type (wt) AKT1 versus the naturally occurring AKT1 mutant (E17K), in human breast cancer. We demonstrated that BPE and HME cells expressing AKT1(E17K) have constitutive plasma membrane localization and hyper-phosphorylation at serine-473 and threonine-308, demonstrating that activation of E17K is growth-factor independent. However, additional studies suggest that HME and BPE cells expressing wt and mutant AKT1 have differing activation kinetics, the reasons of which are unknown. Using a sulforhodamine B and transwell migration assays we demonstrated that E17K inhibits cell growth and migration respectively in BPE and HME cells. However, E17K enhances chemoresistance compared with wt AKT1 function. Interestingly, while E17K inhibits migration in HMLER cells it leads to increased cellular migration in BPLER cells. In order to investigate the molecular basis of AKT1(E17K) function in the different epithelial cell lineages, we performed differential proteomics analysis using liquid chromatography (LC) mass spectrometry (MS) analysis. Although several LCMS methods allowing the characterization and quantification of proteins have been developed we chose two unique styles using 2-dimensional reverse phase reverse phase LC for both. The first method is label-free quantitation utilizing spectral counting. This technique counts the number of spectra identified for a given peptide in each sample and results integrated for all peptides detected for a given protein. The second being iTRAQ, an isobaric labeling technique that uses isotope coded covalent tags for relative quantitation. Using these approaches we identified several hundred differentially expressed proteins between wt and mutant AKT1 and between HME and BPE cells. The proteins from our analysis converged on various cellular functions including pathways underlying cellular migration. Overall the data leads us towards understanding the molecular mechanisms that discriminate between mutant and wt AKT1 function and demonstrates the importance of cellular context in determining genotypic effects of oncogenic mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1060. doi:10.1158/1538-7445.AM2011-1060