Potassium chloride (KCl) has been commonly used in homogenization buffer and procedures of protein extraction. It is known to facilitate release of membrane-associated molecules but the higher concentration of KCl may affect the integrity of mitochondria by breaching the electrostatic force between the lipids and proteins. Therefore, it has been intended to explore the effect of KCl on mitochondrial proteome. The mitochondria were isolated from the mice liver and sub-fractionated into mitochondrial matrix and outer mitochondrial membrane fraction. The fractions were analysed by denaturing polyacrylamide gel electrophoresis (PAGE) and 2D-PAGE. The analysis of ultrastructure and protein profiles by MALDI-MS and data-mining reveals KCl-associated alterations in the integrity of mitochondria and its proteome. The mitochondrial membrane, cristae, and the matrix proteins appear altered under the influence of KCl.

Suman Mishra

Rajnikant Mishra

Molecular Integrity of Mitochondria Alters by Potassium Chloride

Urine has been in the center of attention among scientists of clinical proteomics in the past decade, because it is valuable source of proteins and peptides with a relative stable composition and easy to collect in large and repeated quantities with a noninvasive procedure. In this review, we discuss technical aspects of urinary proteomics in detail, including sample preparation, proteomic technologies, and their advantage and disadvantages. Several recent experiments are presented which applied urinary proteome for biomarker discovery in renal diseases including diabetic nephropathy, immunoglobulin A (IgA) nephropathy, focal segmental glomerulosclerosis, lupus nephritis, membranous nephropathy, and acute kidney injury. In addition, several available databases in urinary proteomics are also briefly introduced.

Shiva Kalantari

Ameneh Jafari

Raheleh Moradpoor

Elmira Ghasemi

Ensieh Khalkhal

Human Urine Proteomics: Analytical Techniques and Clinical Applications in Renal Diseases

Background. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with rapid tumor progression and poor prognosis. This study was motivated by the lack of sensitive and specific PDAC biomarkers and aimed to identify a diagnostic, serum protein signature for PDAC. Methods. To mimic a real life test situation, a multicenter trial comprising a serum sample cohort, including 338 patients with either PDAC or other pancreatic diseases (OPD) and controls with nonpancreatic conditions (NPC), was analyzed on 293-plex recombinant antibody microarrays targeting immunoregulatory and cancer-associated antigens. Results. Serum samples collected from different hospitals were analyzed and showed that (i) sampling from five different hospitals could not be identified as a preanalytical variable and (ii) a multiplexed biomarker signature could be identified, utilizing up to 10 serum markers that could discriminate PDAC from controls, with sensitivities and specificities in the 91&#x2013;100&#x25; range. The first protein profiles associated with the location of the primary tumor in the pancreas could also be identified. Conclusions. The results demonstrate that robust enough serum signatures could be identified in a multicenter trial, potentially contributing to the development of a multiplexed biomarker immunoassay for improved PDAC diagnosis.

Anna S. Gerdtsson

N&#xfa;ria Malats

Anna S&#xe4;ll

Francisco X. Real

Miquel Porta

Petter Skoog

Helena Persson

Christer Wingren

Carl A. K. Borrebaeck

A Multicenter Trial Defining a Serum Protein Signature Associated with Pancreatic Ductal Adenocarcinoma

Hypoxia is an important and common characteristic of many human tumors. It is a challenge clinically due to the correlation with poor prognosis and resistance to radiation and chemotherapy. Understanding the biochemical response to hypoxia would facilitate the development of novel therapeutics for cancer treatment. Here, we investigate alterations in gene expression in response to hypoxia by quantitative proteome analysis using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LCMS/MS. Human HeLa cells were kept either in a hypoxic environment or under normoxic conditions. 125 proteins were found to be regulated, with maximum alteration of 18-fold. In particular, three clusters of differentially regulated proteins were identified, showing significant upregulation of glycolysis and downregulation of mitochondrial ribosomal proteins and translocases. This interaction is likely orchestrated by HIF-1. We also investigated the effect of hypoxia on the cell cycle, which shows accumulation in G1 and a prolonged S phase under these conditions. Implications. This work not only improves our understanding of the response to hypoxia, but also reveals proteins important for malignant progression, which may be targeted in future therapies.

Paula A. Bousquet

Joe Alexander Sandvik

Magnus &#xd8;. Arntzen

Nina F. Jeppesen Edin

Stine Christoffersen

Ute Krengel

Erik O. Pettersen

Bernd Thiede

Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells

Bordetella pertussis (Bp) is the etiologic agent of pertussis (whooping cough), a highly communicable infection. Although pertussis is vaccine preventable, in recent years there has been increased incidence, despite high vaccine coverage. Possible reasons for the rise in cases include the following: Bp strain adaptation, waning vaccine immunity, increased surveillance, and improved clinical diagnostics. A pertussis outbreak impacted California (USA) in 2010; children and preadolescents were the most affected but the burden of disease fell mainly on infants. To identify protein biomarkers associated with this pertussis outbreak, we report a whole cellular protein characterization of six Bp isolates plus the pertussis acellular vaccine strain Bp Tohama I (T), utilizing gel-free proteomics-based mass spectrometry (MS). MS/MS tryptic peptide detection and protein database searching combined with western blot analysis revealed three Bp isolates in this study had markedly reduced detection of pertactin (Prn), a subunit of pertussis acellular vaccines. Additionally, antibody affinity capture technologies were implemented using anti-Bp T rabbit polyclonal antisera and whole cellular proteins to identify putative immunogens. Proteome profiling could shed light on pathogenesis and potentially lay the foundation for reduced infection transmission strategies and improved clinical diagnostics.

Yulanda M. Williamson

Hercules Moura

Jennifer Whitmon

Adrian R. Woolfitt

David M. Schieltz

Jon C. Rees

Stephanie Guo

Heather Kirkham

Daniel Bouck

Edwin W. Ades

Maria Lucia Tondella

George M. Carlone

Jacquelyn S. Sampson

John R. Barr

A Proteomic Characterization of Bordetella pertussis Clinical Isolates Associated with a California State Pertussis Outbreak

Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma, is a heterogeneous disease where the outcome for patients with early relapse or refractory disease is very poor, even in the era of immunochemotherapy. In order to describe possible differences in global protein expression and network patterns, we performed a SILAC-based shotgun (LC-MS/MS) quantitative proteomic analysis in fresh-frozen tumor tissue from two groups of DLBCL patients with totally different clinical outcome: (i) early relapsed or refractory and (ii) long-term progression-free patients. We could identify over 3,500 proteins; more than 1,300 were quantified in all patients and 87 were significantly differentially expressed. By functional annotation analysis on the 66 proteins overexpressed in the progression-free patient group, we found an enrichment of proteins involved in the regulation and organization of the actin cytoskeleton. Also, five proteins from actin cytoskeleton regulation, applied in a supervised regression analysis, could discriminate the two patient groups. In conclusion, SILAC-based shotgun quantitative proteomic analysis appears to be a powerful tool to explore the proteome in DLBCL tumor tissue. Also, as progression-free patients had a higher expression of proteins involved in the actin cytoskeleton protein network, such a pattern indicates a functional role in the sustained response to immunochemotherapy.

Ulla R&#xfc;etschi

Martin Stenson

Sverker Hasselblom

Herman Nilsson-Ehle

Ulrika Hansson

Henrik Fagman

Per-Ola Andersson

SILAC-Based Quantitative Proteomic Analysis of Diffuse Large B-Cell Lymphoma Patients

Even though endoplasmic reticulum (ER) stress associated with mycobacterial infection has been well studied, the molecular basis of ER as a crucial organelle to determine the fate of Mtb is yet to be established. Here, we have studied the ability of Mtb to manipulate the ultrastructural architecture of macrophage ER and found that the ER-phenotypes associated with virulent (H37Rv) and avirulent (H37Ra) strains were different: a rough ER (RER) with the former against a smooth ER (SER) with the later. Further, the functional attributes of these changes were probed by MS-based quantitative proteomics (133 ER proteins) and lipidomics (8 phospholipids). Our omics approaches not only revealed the host pathogen cross-talk but also emphasized how precisely Mtb uses proteins and lipids in combination to give rise to characteristic ER-phenotypes. H37Ra-infected macrophages increased the cytosolic Ca2+ levels by attenuating the ATP2A2 protein and simultaneous induction of PC/PE expression to facilitate apoptosis. However, H37Rv inhibited apoptosis and further controlled the expression of EST-1 and AMRP proteins to disturb cholesterol homeostasis resulting in sustained infection. This approach offers the potential to decipher the specific roles of ER in understanding the cell biology of mycobacterial infection with special reference to the impact of host response.

Najmuddin Mohd Saquib

Shilpa Jamwal

Mukul Kumar Midha

Hirdya Narain Verma

Venkatasamy Manivel

Quantitative Proteomics and Lipidomics Analysis of Endoplasmic Reticulum of Macrophage Infected with Mycobacterium tuberculosis

Objective. Sulfur mustard (SM) is a highly reactive alkylating agent which produces ocular, respiratory, and skin damages. Eyes are the most sensitive organ to SM due to high intrinsic metabolic and rapid turnover rate of corneal epithelium and aqueous-mucous interfaces of the cornea and conjunctiva. Here we investigate underlying molecular mechanism of SM exposure delayed effects which is still a controversial issue after about 30 years. Materials and Methods. Following ethical approval, we have analyzed serum proteome of ten severe SM exposed male patients with delayed eye symptoms with two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. The western blotting was used to confirm the proteins that have been identified. Results. We have identified thirteen proteins including albumin, haptoglobin, and keratin isoforms as well as immunoglobulin kappa chain which showed upregulation while transferrin and alpha 1 antitrypsin revealed downregulation in these patients in comparison with healthy control group. Conclusions. Our results elevated participation of free iron circulatory imbalance and local matrix-metalloproteinase activity in development of delayed ocular symptoms induced by SM. It demonstrates that SM induced systemic toxicity leads to some serum protein changes that continually and gradually exacerbate the ocular surface injuries.

Zaiddodine Pashandi

Neda Saraygord-Afshari

Hossein Naderi-Manesh

Mostafa Naderi

Comparative Proteomic Study Reveals the Molecular Aspects of Delayed Ocular Symptoms Induced by Sulfur Mustard

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International Journal of Proteomics