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Newcastle upon Tyne, United Kingdom

Treumann A.,NEPAF | Thiede B.,University of Oslo
Expert Review of Proteomics | Year: 2010

An important challenge for proteomics is the ability to compare protein levels across biological samples. Since their introduction, isotopic and isobaric peptide labeling have played an important role in relative quantitative comparisons of proteomes. One important drawback of most of the isotopic-labeling techniques is an increase in sample complexity. This problem was successfully addressed with the construction of isobaric labeling strategies, such as isobaric tag for relative and absolute quantification (iTRAQ), tandem mass tagging, the cleavable isobaric affinity tag, dimethylated leucines and isobaric peptide termini labeling. Furthermore, numerous applications for multiplexing using iTRAQ and tandem mass tagging have been reported. © 2010 Expert Reviews Ltd.

Koehler C.J.,University of Oslo | Arntzen M.O.,University of Oslo | Arntzen M.O.,Norwegian University of Life Sciences | Treumann A.,NEPAF | Thiede B.,University of Oslo
Methods in Molecular Biology | Year: 2012

Isobaric peptide termini labeling (IPTL) is a recently introduced approach to the chemical labeling of peptides with isotopic reagents. Peptides derived from two different samples are labeled at the N terminus and at the C terminus with isotopically labeled reagents that have identical mass differences. To obtain isobaric peptides, labeling is carried out such that the introduced mass increase at one terminus will exactly match the mass decrease at the other terminus (and the other way around). This results in product ion spectra that display the quantitative difference of the peptide signal derived from the two samples for every b-ion and y-ion in the spectrum. The original IPTL approach required the selective modification of lysines followed by C-18 micropurification of modified peptides and reaction of the N termini. Here, we describe a new approach for IPTL that is based on the selective modification of the peptide N termini with succinic anhydride and subsequent reductive amination of C-terminal lysines with formaldehyde and cyanoborohydride. Both reactions can be carried out in one pot within 10 min and without C-18 micropurification. In addition, we present the software package IsobariQ for straightforward data analysis. © 2012 Springer Science+Business Media, LLC.

Lai V.K.,University of Leicester | Linares-Palomino J.,University of Leicester | Treumann A.,NEPAF | Saeed M.,University of Leicester | And 3 more authors.
Journal of Surgical Research | Year: 2012

Objectives: The myocardium of patients with diabetes and poor left ventricular (LV) function cannot be protected by interventions such as ischemic preconditioning (IP). We investigated whether these clinical conditions influence the protection elicited by the paracrine effect of bone marrow cells (BMCs) and whether the cause for loss in protection resides in the BMCs, the myocardium, or both. Methods: BMCs and right atrial appendage were obtained from patients with and without diabetes and from poor (EF < 30%) and preserved LV function undergoing elective cardiac surgery. Muscles (n = 6/group) were co-cultured with BMCs and subjected to 90 min ischemia/120 min reoxygenation at 37°C. The degree of protection was assessed by measuring creatine kinase (CK) released, and myocardial cell necrosis and apoptosis. Results: Ischemia-induced CK release, cell necrosis, and apoptosis in the diabetic myocardium were not significantly affected by IP or by co-incubation with autologous or non-diabetic allogenic BMCs. Conversely, significant reduction in CK release, cell necrosis, and apoptosis were observed when non-diabetic myocardium was co-incubated with allogenic diabetic BMCs. Interestingly, while allogenic BMCs from subjects with preserved LV function exerted a modest but significant reduction in CK leakage and cell necrosis, but not apoptosis, on failing myocardium, the BMCs from patients with poor LV function failed to protect their own and the allogenic myocardium from subjects with normal LV function. Conclusions: The failure to protect the myocardium of patients with poor LV function against ischemia/reoxygenation-induced injury is mainly due to a deficit in their BMCs and the myocardium itself, whereas in patients with diabetes the deficit remains within the myocardium and not in the BMCs. © 2012 Elsevier Inc. All rights reserved.

Koehler C.J.,University of Oslo | Arntzen M.O.,University of Oslo | Treumann A.,NEPAF | Thiede B.,University of Oslo
Analytical and Bioanalytical Chemistry | Year: 2012

Isobaric peptide termini labeling (IPTL) is a quantification method which permits relative quantification using quantification points distributed throughout the whole tandem mass spectrometry (MS/MS) spectrum. It is based on the complementary derivatization of peptide termini with different isotopes resulting in isobaric peptides. Here, we use our recently developed software package IsobariQ to investigate how processing and data analysis parameters can improve IPTL data. Deisotoping provided cleaner MS/MS spectra and improved protein identification and quantification. Denoising should be used with caution because it may remove highly regulated ion pairs. An outlier detection algorithm on the ratios within every individual MS/MS spectrum was beneficial in removing false-positive quantification points. MS/MS spectra using IPTL typically contain two peptide series with complementary labels resulting in lower Mascot ion scores than non-labeled equivalent peptides. To avoid this penalty, the two chemical modifications for IPTL were specified as variables including satellite neutral losses of tetradeuterium with positive loss for the heavy isotopes and negative loss for the light isotopes. Thus, the less dominant complementary ion series were not considered for the scoring, which improved the ion scores significantly. In addition, we showed that IPTL was suitable for fragmentation by electron transfer dissociation (ETD) and higher energy collisionally activated dissociation (HCD) besides the already reported collision-induced dissociation (CID). Notably, ETD and HCD data can be identified and quantified using IsobariQ. ETD outperformed CID and HCD only for charge states ≥4+ but yielded in total fewer protein identifications and quantifications. In contrast, the high-resolution information of HCD fragmented peptides provided most identification and quantification results using the same scan speed. © 2012 Springer-Verlag.

Thiede B.,University of Oslo | Koehler C.J.,University of Oslo | Strozynski M.,University of Oslo | Treumann A.,NEPAF | And 4 more authors.
Molecular and Cellular Proteomics | Year: 2013

The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as hown in detail for lamin A/C and vimentin, the quantiative changes of protein species can be detected. The ombination of 2-DE with high-resolution nano-liquid hromatography-mass spectrometry allowed us to idenify proteomic changes in apoptotic cells that would be nobservable using any of the other previously emloyed proteomic workflows. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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