The design and synthesis of a proline-based reporter isobaric Tandem Mass Tag structure (TMTpro) is presented. An analysis is made of the performance of the new TMTpro tags in comparison with the current commercially available dimethylpiperidine-reporter-based TMT10/11 reagents. The new reporter structure provides a set of 16 tags for use with resolution of 6.3 mDa mass differences in high resolution mass spectrometers and a set of 9 reagents with 1 Da spacing between reporter ions for single dalton analysis using 9 heavy nuclei per tag. We show similar performance in terms of peptide identification rates and quantification between the TMTpro 16-plex and TMT10/11-plex reagents. We also demonstrate the suitability of the TMTpro reagents for phosphopeptide analysis. The ability to pool 16 samples reduces the overall amount of sample required for each channel, and we anticipate that TMTpro reagents will be a useful enhancement for any protocol that benefits from sample pooling and should reduce missing data.

Objective-Polymorphonuclear neutrophils (PMNs) play a main role in abdominal aortic aneurysm (AAA) progression.We have analyzed circulating PMNs isolated from AAA patients and controls by a proteomic approach to identify proteins potentially involved in AAA pathogenesis. Methods and Results-PMNs from 8 AAA patients (4 large AAA Ͼ5 cm and 4 small AAA 3-5 cm) and 4 controls were analyzed by 2D differential in-gel electrophoresis. Among differentially expressed spots, several proteins involved in redox balance were identified by mass spectrometry (eg, cyclophilin, thioredoxin reductase, catalase). Diminished catalase expression and activity were observed in PMNs from AAA patients compared with controls. In contrast, PMNs from AAA patients displayed higher H 2 O 2 and myeloperoxidase levels than PMNs from controls. Moreover, a significant decrease in catalase mRNA levels was observed in PMNs after phorbol 12-myristate 13-acetate incubation. Catalase plasma levels were also decreased in large (nϭ47) and small (nϭ56) AAA patients compared with controls (nϭ34). We observed catalase expression in AAA thrombus and thrombus-conditioned medium, associated with PMN infiltration. Furthermore, increased H 2 O 2 levels were observed in AAA thrombus-conditioned medium compared with the media layer. Key Words: aneurysms Ⅲ antioxidants Ⅲ leukocytes A bdominal aortic aneurysm (AAA) is an important health problem in elderly. In cross-sectional studies, the prevalence varies from 3% to 8%. 1 In elderly men, AAAs may cause as much as 2% to 3% of all deaths. 1 Because AAAs are usually asymptomatic, the present clinical challenges are early diagnosis and deciphering the biological mechanisms responsible for the progressive dilatation and final rupture to develop new diagnostic and therapeutic approaches. Conclusion-DiminishedAlthough polymorphonuclear neutrophils (PMNs) represent the major class of leukocytes, they have received little attention in atherothrombosis. 2,3 However, recent evidence is revealing a previously unappreciated role of PMN in experimental 4,5 and human 6,7 AAAs. PMNs can contribute to main mechanisms of AAA evolution, namely intraluminal thrombus (ILT) formation, oxidative stress, proteolytic degradation of the aortic media, and adventitial inflammation. 8 AAAs are characterized by the presence of a mural ILT-containing platelets, red blood cells (RBCs), and PMNs, particularly abundant within the luminal layer of human thrombus. 6 -8 AAAs are also characterized by destructive connective tissue remodeling, including depletion of aortic elastin and fragmentation of medial elastic fibers. 9 Finally, inflammatory cells (macrophages and neutrophils) are also evident within the adventitia of human AAAs. 10 Interestingly, PMNs depletion is able to inhibit experimental AAA formation. 11 More recently, short-term preoperative doxycycline therapy improved the proteolytic balance in human AAA, presumably via an effect on aortic wall neutrophil content. 12 These data highlight the potential interest of analyzing the PMNs p...

Objective-In the search of novel biomarkers of abdominal aortic aneurysm (AAA) progression, proteins released by intraluminal thrombus (ILT) were analyzed by a differential proteomic approach. Methods and Results-Different layers (luminal/abluminal) of the ILT of AAA were incubated, and the proteins released were analyzed by 2-dimensional difference in-gel electrophoresis. Several differentially expressed proteins involved in main AAA pathological mechanisms (proteolysis, oxidative stress, and thrombosis) were identified by mass spectrometry. Among the proteins identified, peroxiredoxin-1 (PRX-1) was more released by the luminal layer compared with the abluminal layer of the ILT, which was further validated by Western blot, ELISA, and immunohistochemistry. We demonstrated increased PRX-1 serum levels in AAA patients compared with healthy subjects and also positive correlation among PRX-1 and AAA diameter, plasmin-antiplasmin, and myeloperoxidase levels. Finally, a prospective study revealed a positive correlation between PRX-1 serum levels and AAA expansion rate. Moreover, the combination of PRX-1 and AAA size had significantly additive value in predicting growth. is an important health problem, which occurs in up to 9% of adults older than 65 years of age. The incidence of asymptomatic and ruptured AAA has increased during recent decades, causing Ϸ1% to 2% of male deaths in Western countries. 1 Because AAAs are usually asymptomatic before rupture, the present clinical challenges are to diagnose AAA at an early stage and to decipher the biological mechanisms leading to progressive dilatation and finally rupture, to develop new diagnostic and therapeutic approaches. Identification of biomarkers could help to target both objectives. Conclusion-SeveralPrevious studies have identified AAA biomarkers by studying the levels of different molecules potentially related to AAA pathological mechanisms. 2,3 A different noncandidate biomarker strategy using a set of modern high-throughput technologies, including proteomics, will offer new opportunities to gain a deeper insight into disease processes, including their molecular mechanisms, the risk factors involved, and the analysis of disease progression. 4,5 We have previously reported a differential proteomic approach to identify new atherothrombosis biomarkers released by the arterial wall into plasma using normal and pathological arteries in culture. 6,7 The presence of an intraluminal thrombus (ILT) is a main feature of AAA, and recent data suggest that the biological activities of ILT play a major role in AAA development in humans. 8 In this study, different layers (luminal/abluminal) from the ILT of human AAA patients were incubated in protein-free medium, and the released proteins were analyzed by a gel-based (2-dimensional difference in-gel electrophoresis [2D-DIGE]) proteomic approach followed by protein identification by mass spectrometry (MS). We focused on peroxiredoxin-1 (PRX-1) in view of its potential role in modulating oxidative stress and thus validated its ...

Objective-To identify proteins related to intraluminal thrombus biological activities that could help to find novel pathological mechanisms and therapeutic targets for human abdominal aortic aneurysm (AAA). Approach and Results-Tissue-conditioned media from patients with AAA were analyzed by a mass spectrometry-based strategy using liquid chromatography coupled to tandem mass spectrometry. Global pathway analysis by Ingenuity software highlighted the presence of several circulating proteins, among them were proteins from the complement system. Complement C3 concentration and activation were assessed in plasma from AAA patients (small AAA, AAA diameter=3-5 cm and large AAA, AAA diameter >5 cm), showing decreased C3 levels and activation in large AAA patients. No association of a combination of single-nucleotide polymorphisms in complement genes between large and small AAA patients was observed. Intense extracellular C3 inmunostaining, along with C9, was observed in AAA thrombus. Analysis of C3 in AAA tissue homogenates and tissue-conditioned media showed increased levels of C3 in AAA thrombus, as well as proteolytic fragments (C3a/C3c/C3dg), suggesting its local deposition and activation. Finally, the functional role of local complement activation in polymorphonuclear (PMN) cell activation was tested, showing that C3 blockade by anti-C3 antibody was able to decrease thrombus-induced neutrophil chemotaxis and reactive oxygen species production. Conclusions-

Previous studies have shown that oxidized products of the phospholipid PAPC (Ox-PAPC) are strong activators of aortic endothelial cells and play an important role in atherosclerosis and other inflammatory diseases. We and others have demonstrated that Ox-PAPC activates specific signaling pathways and regulates a large number of genes. Using a phosphoproteomic approach based on phosphopeptide enrichment and mass spectrometry analysis, we identified candidate changes in Ox-PAPC-induced protein phosphorylation of 228 proteins. Functional annotation of these proteins showed an enrichment of the regulation of cytoskeleton, junctional components, and tyrosine kinases, all of which may contribute to the phenotypic and molecular changes observed in endothelial cells treated with Ox-PAPC. Many changes in protein phosphorylation induced by Ox-PAPC are reported here for the first time and provide new insights into the mechanism of activation by oxidized lipids, including phosphorylation-based signal transduction.