Cover Picture: Proteomics – Clinical Applications 9/2008

In this issue of Proteomics – Clinical Applications you will find the following highlighted articles: Always probing for more: prostate biomarkers It feels a bit like the late nineteenth century, but instead of a gold rush every two to five years, it's a new favorite target in the biomarker rushes. (Actually, gold rushes go back to ancient Egypt. Biomarkers don't go that far but medical research does.) Here, Burgess et al. take a walk outside the box when they encounter the asymmetry of protein abundance. Rather than synthetically trapping compounds to expose or capture low abundance compounds, they use nature's own: in particular, alpha‐2‐macroglobulin (A2M). A2Ms normal function is to bind proteins that are to be protected from proteolysis, a universal protease inhibitor. Using immunoprecipitation of A2M and comparing cases vs. controls, enhanced levels of heat shock protein 90 in serum was their most interesting candidate for this year's marker rush. Burgess, E. F. et al., Proteomics Clin. Appl. 2008, 2, 1223–1233. Brainwashing samples No, we are not suggesting 1984‐style re‐education to improve proteome productivity. Rather, Dean et al. are reporting on the efficiency of fractionation of brain tissue proteins by graduated detergent extraction prior to 2‐DE. Another anticipated benefit is increased relative concentration of the less abundant proteins. Samples from two areas of the human brain (Brodmann's Area 9 (BA9) and caudate nucleus and putamen CP) were prepared with a sequential extraction kit and compared by 1‐DE and Western blots, 2‐DE and MALDI‐TOF. The conclusion was that no detergent conditions were found that resolved proteins completely but that each detergent point gave a different 2‐D pattern, a benefit for those looking for distinguishing marks. Dean, B. et al., Proteomics Clin. Appl. 2008, 2, 1281–1289. Liver and kidney pie In orthotopic (“full replacement”) liver transplants, one of the most common complications is chronic kidney (yes, kidney) disease. Currently, kidney complications are tracked by functional tests, like serum urea and creatinine levels. If things look suspicious, glomerular filtration rates can be checked. O'Riordan et al. applied SELDI TOF‐MS techniques to serum samples to look for easier, more accurate targets. Serum samples were collected repeatedly over a 6‐month period. Each was divided into six fractions by elution pH or by organic solvent, then examined on weak cation exchange (CM10), hydrophobic (H50) and immobilized metal affinity surfaces (IMAC30). CM10 was best at distinguishing case from control using three proteins and reporting a sensitivity of ～87–94%. On the basis of peptide LC‐MS and 1‐D SDS‐PAGE and confirmed by ELISA, the best single indicator was APO‐AI. Most cases of kidney disease appeared to be linked to the use of calcineurin inhibitors for immune suppression. O'Riordan, A. et al., Proteomics Clin. Appl. 2008, 2, 1338–1348.     

Cover Picture: Proteomics – Clinical Applications 1/2008

In this issue of Proteomics you will find the following highlighted articles: Colon Cancer Complements to 2‐DE from 2‐D‐LC “When you have a good thing going, run with it” – Quote from paleolithic philosopher and hunting consultant. In this case, Thierolf et al. took a colon cancer sample set well‐characterized by 2‐DE‐MALDI PMF and ran it through a 2‐D‐LC‐ESI‐MS protocol. The samples of malignant and normal tissues from the same patient, analyzed by 2‐DE‐MALDI and mass fingerprinting (reported elsewhere) yielded 734 unique proteins. When the same tissue specimens were analyzed by 2‐D‐LC‐ESI‐MS, 484 proteins were identified, 232 of them new and 252 that had been ID’d in the earlier work. The two unique sets exhibited similar functional and ontological profiles, confirming the complementarity of the two methods. Using the data to select up‐regulated proteins for evaluation of potential for serving as biomarkers, they chose S100A12 as particularly interesting. An ELISA found that it was more sensitive but less discriminating than carcinoembryonic antigen. S100A12 is also an inflammation marker. Thierolf, M. et al., Proteomics Clin. Appl. 2008, 2, 11–22 Urinary bladder cancer: A proteomic approach Standing at number five on the US cancer frequency list, urinary bladder cancer costs almost $3 billion a year. No acceptable early detection tests have been developed – it seems no one will volunteer for a “routine” annual cystoscopy, so the 5‐year survival rate has stayed below 50% for many years. Several urinary biomarkers have been developed but fall short in their frequency of false positives or false negatives. Using 2‐DE/MALDI‐TOF MS and Ingenuity Systems’ Pathway Analysis software, Li et al. surveyed invasive urothelial carcinomas for up‐regulated proteins and settled on Choro­ideremia‐like protein (CHML) out of 21 candidates. Immunohistochemistry and Western blotting verified the specificity. The functional pathways found are part of the lipid metabolism, inflammation and molecular transport machinery and CHML is part of the Rab geranylgeranylation pathway. More work is needed to understand the diagnostic potential of CHML. Li, J. et al., Proteomics Clin. Appl. 2008, 2, 78–89. Angina: Looking for markers in all the right places Angina, the chest pain associated with heart attacks, has two forms: stable (SAP) and unstable (UAP). SAP is relieved by rest. UAP pain persists at rest and is often due to formation of a clot which can lead to major or fatal damage (ischemia, myocardial infarct). The ability to distinguish the two would be a boon to hospital emergency care facilities because admission and intensive care are not required for SAP. Brown et al. report here the use of anti‐leukocyte antibody arrays to analyze circulating cells by surface CD antigen type. Starting with 82 antibodies, they could readily distinguish healthy from SAP and UAP cases from 8 and 19 spot intensity differences, respectively. SAP and UAP could be separated with seven markers using spot intensity and cluster analysis, but not as cleanly, possibly because SAP has a tendency to convert to UAP. Brown, A. et al., Proteomics Clin. Appl. 2008, 2, 90–98.     

Cover Picture: Proteomics – Clinical Applications 12/2008

In this issue of Proteomics – Clinical Applications you will find the following highlighted articles: Looking through the leftovers The magic in conditioned medium has been recognized for decades but exactly what it was and how it worked has only begun to come to light more recently. Byproducts of metabolic activity are easy to spot, more challenging are the secreted growth factors and other types of communication molecules. Ogura et al. looked at the litter that surrounded various cancer cells as a potential gold mine. It took only a bit of RP‐HPLC and MALDI‐TOF panning to turn up nuggets of pro‐ and pre‐proneurotensin/neuromedin N (pre‐proNT/NTN), good candidates for small‐cell lung cancer biomarkers. Pre‐proNT/NTN was found in medium from 4 out of 7 different small‐cell lung carcinomas but 0 out of 8 non‐small‐cell carcinomas. Ogura, S.‐i. et al., Proteomics Clin. Appl. 2008, 2, 1620–1627. Hunting for Huntington's Creating animal models of inherited human diseases is not always a simple issue of replacing an animal gene with the human equivalent. Huntington's Disease (HD), caused at least in part by the development of expanded polyglutamine (CAG)n sequences in the huntingtin gene, has been modeled in mice with n>60 (CAG)n sequences but this leads to expression as a juvenile form of the disease. Nguyen et al. have developed transgenic rats that come much closer to the pattern of adult human HD in the character and time of appearance of motor deficits, cognitive decline, and emotional disturbance. After thorough micro array evaluation of the rat model at ages 3 and 12 months, these researchers argue that they are much closer to a system suitable for selection and application of biomarkers and potential therapeutics. Nguyen, H. P. et al., Proteomics Clin. Appl. 2008, 2, 1638–1650. Bifunctional assay means less work To be able to have your cake and eat it, too, is one of those things economists tell us is impossible. Don't tell Rader et al. though. They are investigating methods to simplify and speed up human papilloma virus (HPV) biomarker screening from the same sample. This would be very useful for cervical cancer staging, still a problem despite the recent introduction and growing use of a vaccine for the most frequent cancer‐causing HPV types. Cervical samples were collected into a tube containing an RNA stabilizing reagent, from which proteins could be extracted and freed of cervical mucus. RNA could be extracted from the same samples with Trizol reagent according to the manufacturer's directions. Proteins recovered were cleanly analyzed by 2‐D DIGE and Western blots; total RNA could be analyzed on human cDNA arrays. Rader, J. S. et al., Proteomics Clin. Appl. 2008, 2, 1658–1669.