EuPA Open Proteomics最新文献

This work on solving the mystery of words encoded by amino acids in peptides was derived by the YPIC-EuPA Challenge. We received a dry synthetic peptide sample and performed a mass spectrometric analysis followed by de novo peptide sequencing. As a result, a part of “Rays of positive electricity and their application to chemical analyses” by J.J.Tomson was found to be encoded in the peptides of the sample. The words were first revealed from the peptides, that matched by Google search to find the answer. After that, the answer was validated using a standard proteomic search against a database constructed from the quotation found.

Using primarily LC-MS/MS de novo sequencing techniques, we concluded that the peptides form the following sentence from Sir JJ Thomson’s preface to Rays of Positive Electricity and Their Application to Chemical Analyses:

“I feel sure that there are many problems in chemistry which could be solved with far greater ease by this than by any other method. The method is surprisingly sensitive, more so even than that of spectrum analysis, requires an infinitesimal amount of material, and does not require this to be specially purified.”

Here, we detail our process for deciphering the peptide mixture composition and finding the sentence they form and from what book the sentence comes from.

Here we present the results of our attempt on the EuPA YPIC challenge. The task was to sequence the provided synthetic peptides, build the sentence encrypted in them and determine from which book the sentence originated.

The task itself, while holding no direct scientific value, offers an insight in less formal terms (for participants at least) on how the overall process of a scientific study of a “new protein” looks like. Hence, we decided to look at the challenge as if it was a general task of sequencing an unknown protein from an unusual proteome database. To solve the task we used LC–MS/MS, MALDI-MS and de novo sequencing. A combination of two MS instruments and de novo MS/MS data analysis make it possible to sequence new peptides and proteins not yet present in proteomic databases.

Glucose Oxidase (GOD), is a common flavoprotein from Aspergillus niger ATCC 9029 with a broad application in biotechnology, food and medical industries. In this study, GOD gene was cloned into the expression vector, pPIC9 and screened by the alcohol oxidase promoter. The enzyme production increased at 28 °C. GOD activity induced by 1.0% methanol and the highest level of GOD production was the result of shaking rate at 225 rpm. The highest enzyme activity obtained at a pH value ranged from 5 to 7 at 50 °C. The enzyme was stable at a broad pH range and temperature.

We previously characterized Pga1, a Candida albicans (C. albicans) cell wall protein necessary for proper virulence, adhesion, and resistance to oxidative stress. By utilizing tandem mass spectrometry coupled with bioinformatics to investigate cell wall proteome expression in a pga1 null fourteen and 36 proteins were identified in the wild type grown under filamentous and non-filamentous conditions respectively, but were not detected in the mutant, including members of the PGA GPI anchored family. Virulence and adhesion proteins such as Hsp 90, Sap10, Cdc11, Int 3 and members of the lipase family were also identified exclusively in the wild type.

Introduction

Fine Needle Aspiration Biopsy (FNAB) allows the cytological differentiation of benign and malignant thyroid nodules. However, the method itself is not adequate in determining some cases. For example, the diagnosis of Follicular Variant Papillary Thyroid Carcinoma (FV-PTC) can be challenging. In the current study we investigate the protein profiles of FV-PTC and classical variant PTC (CV-PTC) with no lymph node metastasis and compare it with benign thyroid tissue.

Method

We used CV-PTC (n = 6), FV-PTC (n = 6) and benign thyroid tissues (n = 6) to prepare tissue lysates. Proteins from each group were trypsin and lys-C digested. The samples were analyzed on a Q Exactive Orbitrap mass spectrometer.

Results

We identified 2560 proteins across all 18 specimens. Protein profiles revealed that there was no clear distinction between benign and FV-PTC samples. However, further examination of our data showed that proteins in energy metabolism have altered in FV-PTC. Proteomic pathway analysis showed marked alteration of the actin cytoskeleton proteins, especially several members of Arp2/3 complex were significantly increased in CV-PTC. We made the novel observation that IQGAP1 protein was significantly increased in CV-PTC, whereas IQGAP2 protein was highly expressed in FV-PTC lesions, suggesting differential roles of IQGAP proteins in thyroid pathology.

Conclusion

In the present study, mass spectrometry based label free quantification approach was applied to investigate the protein profiles of FV-PTC, CV-PTC and benign thyroid tissues. This study pointed out that actin cytoskeleton proteins, IQGAP proteins and changes in energy metabolism play predominant roles in thyroid pathology.

SH-SY5Y neuroblastoma cells are susceptible to differentiation using retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), providing a model of neuronal differentiation. We compared SH-SY5Y cells proteome before and after RA/BDNF treatment using iTRAQ and phosphopeptide enrichment strategies. We identified 5587 proteins, 366 of them with differential abundance. Differentiated cells expressed proteins related to neuronal development, and, undifferentiated cells expressed proteins involved in cell proliferation. Interactive network covered focal adhesion, cytoskeleton dynamics and neurodegenerative diseases processes and regulation of mitogen-activated protein kinase-related signaling pathways; key proteins involved in those processes might be explored as markers for neuronal differentiation.

Peptidase inhibitors have an important role controlling a variety of biological processes. Here, we employed a peptidomic approach including molecular cloning, tandem mass spectrometry and enzymatic assays to reveal 7 Kazal-type proteinase inhibitors (CCKPs) (18 variants) in the skin secretion of the unexplored frog, Cruziohyla calcarifer. All 18 proteins shared the Kazal pattern C-X₍₇₎-C-X_(6,7)-C-X_(6,7)-Y-X₍₃₎-C-X₍₂₎-C-X_(15-21)-C and 3 disulphide bridges. Based on structural comparative analysis, we deemed trypsin and chymotrypsin inhibitory activity in CCKP-1, 4 and CCKP 2, 5, 7, respectively. These peptidase inhibitors presumably play a role to control the balance between other functional peptides produced in the amphibian skin secretions.