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 4/2008

In this issue of Proteomics you will find the following highlighted articles: MALDI and methyl groups take on lysosomal storage disease diagnosis Lysosomal storage disorders (LSD) result from the absence or loss of any of a wide variety of glycan‐processing enzymes that lead to accumulation of incompletely processed glyco‐molecules in lysosomes. Diagnosis of the particular type of LSD requires identification of the accumulated species, usually from urine. LSD diagnosis has been a target for many years, including use of GC/MS, TLC, NMR, HPLC, FACE and other techniques. Faid et al. take a simple approach that is quite successful and much less time consuming. After permethylating crude urine, a sample is first run on GC/MS to look for free sialic acid, an indicator of one set of diseases, then a cleaned up sample (C_1–8) is analyzed by MALDI/TOF to characterize glycoproteins and glycopeptides present. The glycans were further analyzed by sequential exoglucosidase digestion on the MALDI target. Sulfated glycocompound analysis requires a desulfation step prior to permethylation. Faid, V. et al., Proteomics Clin. Appl. 2008, 2, 528–542. Popular prostate problem receives piercing look Prostate cancer (PCa) is one of the most frequent male cancers. It is also one of the most frequently misdiagnosed cancers. Prostate specific antigen (PSA) has improved the accuracy somewhat but still generates 700 000 false positives requiring biopsies per year in the US. And very low PSA levels don’t assure freedom from PCa. In short, there is no good biomarker for PCa. Theodorescu et al. recognized that anything recovered from blood had a good chance of being at least partially degraded so they looked at first void urine as a more likely source of stable material. Applying capillary electrophoresis coupled to TOF‐MS, the authors developed an “informative” panel of eight peptides that distinguished between first void and mid‐stream samples and a PCa‐specific panel of 12 proteins that detected the disease. Incorporating the 12 proteins with age and free PSA, sensitivity was 91%, specificity 69%. Theodorescu, D. et al., Proteomics Clin. Appl. 2008, 2, 556–570. No candy in candidiasis Candida albicans is a unicellular yeast that is the third most common nosocomial agent in ICU’s, costing ～US$ 40 000 per incident. Early diagnosis of systemic candidiasis (SC) is crucial for successful control of the infection by antifungal therapies. Blood cultures are not fast and tissue biopsies are not sensitive but are the current “gold standards.” Pitarch et al. report here a serum protein marker for SC: anti‐Candida enolase (Eno1p) IgG. Comparing healthy with SC patients for antibodies against Candida cytoplasmic antigens, 15 antigens were recognized by the SC patients. The strongest response was to Eno1p which was the only antigen to produce a response in all 12 patients. The IgG was tested for its suitability as an antigen in Western and capture ELISA tests. Although the tests appear reliable for detecting SC, they were not good for prognosis. A considerable amount of work remains for full qualification of the test. Pitarch, A. et al., Proteomics Clin. Appl. 2008, 2, 596–618.     

In this issue: 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.     

In this issue: Proteomics – Clinical Applications 4/2008

In this issue of Proteomics you will find the following highlighted articles: MALDI and methyl groups take on lysosomal storage disease diagnosis Lysosomal storage disorders (LSD) result from the absence or loss of any of a wide variety of glycan‐processing enzymes that lead to accumulation of incompletely processed glyco‐molecules in lysosomes. Diagnosis of the particular type of LSD requires identification of the accumulated species, usually from urine. LSD diagnosis has been a target for many years, including use of GC/MS, TLC, NMR, HPLC, FACE and other techniques. Faid et al. take a simple approach that is quite successful and much less time consuming. After permethylating crude urine, a sample is first run on GC/MS to look for free sialic acid, an indicator of one set of diseases, then a cleaned up sample (C_1–8) is analyzed by MALDI/TOF to characterize glycoproteins and glycopeptides present. The glycans were further analyzed by sequential exoglucosidase digestion on the MALDI target. Sulfated glycocompound analysis requires a desulfation step prior to permethylation. Faid, V. et al., Proteomics Clin. Appl. 2008, 2, 528–542. Popular prostate problem receives piercing look Prostate cancer (PCa) is one of the most frequent male cancers. It is also one of the most frequently misdiagnosed cancers. Prostate specific antigen (PSA) has improved the accuracy somewhat but still generates 700 000 false positives requiring biopsies per year in the US. And very low PSA levels don’t assure freedom from PCa. In short, there is no good biomarker for PCa. Theodorescu et al. recognized that anything recovered from blood had a good chance of being at least partially degraded so they looked at first void urine as a more likely source of stable material. Applying capillary electrophoresis coupled to TOF‐MS, the authors developed an “informative” panel of eight peptides that distinguished between first void and mid‐stream samples and a PCa‐specific panel of 12 proteins that detected the disease. Incorporating the 12 proteins with age and free PSA, sensitivity was 91%, specificity 69%. Theodorescu, D. et al., Proteomics Clin. Appl. 2008, 2, 556–570. No candy in candidiasis Candida albicans is a unicellular yeast that is the third most common nosocomial agent in ICU’s, costing ～US$ 40 000 per incident. Early diagnosis of systemic candidiasis (SC) is crucial for successful control of the infection by antifungal therapies. Blood cultures are not fast and tissue biopsies are not sensitive but are the current “gold standards.” Pitarch et al. report here a serum protein marker for SC: anti‐Candida enolase (Eno1p) IgG. Comparing healthy with SC patients for antibodies against Candida cytoplasmic antigens, 15 antigens were recognized by the SC patients. The strongest response was to Eno1p which was the only antigen to produce a response in all 12 patients. The IgG was tested for its suitability as an antigen in Western and capture ELISA tests. Although the tests appear reliable for detecting SC, they were not good for prognosis. A considerable amount of work remains for full qualification of the test. Pitarch, A. et al., Proteomics Clin. Appl. 2008, 2, 596–618.     

In this issue: 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 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.     

In this issue: 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.     

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.     

Towards a comprehensive proteome of normal and malignant human colon tissue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.     

Evaluation of a type 1 diabetes serum cohort by SELDI-TOF MS protein profiling

Proteomics analysis of serum from patients with type 1 diabetes (T1D) may lead to novel biomarkers for prediction of disease and for patient monitoring. However, the serum proteome is highly sensitive to sample processing and before proteomics biomarker research serum cohorts should preferably be examined for potential bias between sample groups. SELDI‐TOF MS protein profiling was used for preliminary evaluation of a biological‐bank with 766 serum samples from 270 patients with T1D, collected at 18 different paediatric centers representing 15 countries in Europe and Japan over 2 years (2000–2002). Samples collected 1 (n = 270), 6 (n = 248), and 12 (n = 248) months after T1D diagnosis were grouped across centers and compared. The serum protein profiles varied with collection site and day of analysis; however, markers of sample processing were not systematically different between samples collected at different times after diagnosis. Three members of the apolipoprotein family increased with time in patient serum collected 1, 6, and 12 months after diagnosis (ANOVA, p<0.001). These results support the use of this serum cohort for further proteomic studies and illustrate the potential of high‐throughput MALDI/SELDI‐TOF MS protein profiling for evaluation of serum cohorts before proteomics biomarker research.     

Assessment by Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Spectrometry of the Effects of Preanalytical Variables on Serum Peptidome Profiles Following Long‐Term Sample Storage

Purpose

Human serum and plasma are often used as clinical specimens in proteomics analyses, and peptidome profiling of human serum is a promising tool for identifying novel disease‐associated biomarkers. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is widely used for peptidomic biomarker discovery. Careful sample collection and handling are required as either can have a profound impact on serum peptidome patterns, yet the effects of preanalytical variables on serum peptidome profiles have not been completely elucidated. The present study investigated the effects of preanalytical variables, including storage temperature, duration (up to 12 months), and thawing methods, on MALDI‐TOF MS‐based serum peptidome patterns.

Experimental design

Aliquots of serum samples were pretreated with weak cation exchanger magnetic beads using an automated ClinProtRobot system and then analyzed by MALDI‐TOF MS.

Results

A number of significant differences in peak intensities were observed depending on sample processing variables.

Conclusions and clinical relevance

These peaks can be used as sample quality markers to assess the effects of long‐term storage on serum peptidome profiles using MALDI‐TOF MS.

     

The future of targeted peptidomics

Targeted MS is becoming increasingly important for sensitive and specific quantitative detection of proteins and respective PTMs. In this article, Ceglarek et al. [Proteomics Clin. Appl. 2013, 7, 794–801] present an LC-MS-based method for simultaneous quantitation of seven apolipoproteins in serum specimens. The assay fulfills many necessities of routine diagnostic applications, namely, low cost, high throughput, and good reproducibility. We anticipate that validation of new biomarkers will speed up with this technology and the palette of laboratory-based diagnostic tools will hopefully be augmented significantly in the near future.     

Translating N‐Glycan Analytical Applications into Clinical Strategies for Ovarian Cancer

Protein glycosylation, particularly N‐linked glycosylation, is a complex posttranslational modification (PTM), which plays an important role in protein folding and conformation, regulating protein stability and activity, cell–cell interaction, and cell signaling pathways. This review focuses on analytical techniques, primarily MS‐based techniques, to qualitatively and quantitatively assess N‐glycosylation while successfully characterizing compositional, structural, and linkage features with high specificity and sensitivity. The analytical techniques explored in this review include LC–ESI–MS/MS and MALDI time‐of‐flight MS (MALDI‐TOF‐MS), which have been used to analyze clinical samples, such as serum, plasma, ascites, and tissue. Targeting the aberrant N‐glycosylation patterns observed in MALDI–MS imaging (MSI) offers a platform to visualize N‐glycans in tissue‐specific regions. The studies on the intra‐patient (i.e., a comparison of tissue‐specific regions from the same patient) and inter‐patient (i.e., a comparison of tissue‐specific regions between different patients) variation of early‐ and late‐stage ovarian cancer (OC) patients identify specific N‐glycan differences that improve understanding of the tumor microenvironment and potentially improve therapeutic strategies for the clinic.     

Using fibers for rapid extraction of proteins from urine

The method for rapid extraction of proteins from urine published in this special issue (Manard, B. T. et al., Proteomics Clin. Appl. 2015, 9, 522–530) may have more profound implications than the authors have claimed, simply because urine is more important than most biomarker researchers realized. Unlike blood that is tightly controlled by homeostatic mechanisms of the body, urine tolerates, and accumulates a much larger degree of changes in its components, making it a more important biomarker source than blood.     

Heavy hitting: Using water to label humans

Monitoring protein dynamics, compared to measuring static protein expression profiles taken with snapshot evaluations, have recently been the focus of proteomics studies examining tissue or blood samples where time course changes occur. Using deuterium oxide (²H₂O) to label amino acids is a useful method to monitor protein turnover rates. The synthesis rate for individual proteins is calculated from the rate of ²H incorporation into specific proteins analyzed by high resolution MS. In this issue, Wang and colleagues measured the plasma protein turnover dynamics in healthy humans by in vivo ²H₂O labeling [Wang, D. et al., Proteomics Clin. Appl. 2014, 8, 610–619]. The authors developed and validated a safe and accessible ²H₂O administration protocol to record the turnover dynamics of 542 plasma proteins using MS. Their study demonstrates a promising new way to evaluate plasma protein dynamics in clinical trials where such knowledge could help for prognosis and evaluating treatment efficacy.     

Identification of melanoma‐specific serological markers using proteomic analyses

The discovery of novel melanoma markers for not only early detection but also monitoring disease status is promising to improve the clinical outcome of patients. In the present study, we performed proteomic comparative analysis of plasma proteins between healthy volunteers and melanoma patients using NanoLC and MALDI‐TOF‐MS. As a result, we were successful in identifying nine proteins that were specifically expressed in melanoma plasma compared with healthy plasma, most of which had not previously been identified as plasma markers of melanoma. The mRNA expression levels of four proteins [pro‐platelet basic protein precursor (PPBP), serum amyloid A2 (SAA2), complement factor H‐related protein 1 precursor (FHR1), inter‐alpha‐trypsin inhibitor heavy chain H4 precursor (IAIH4)] were prominently up‐regulated in several melanoma cell lines compared with melanocytes. Moreover, two proteins (PPBP, SAA) were shown to be expressed in tumor specimens from melanoma patients. In the survival time analysis regarding melanoma patients, the semi‐quantified plasma PPBP levels were statistically negatively correlated with the survival time. Most interestingly, the significant survival benefit was seen in low PBPP level group (< index 20) versus high level (≥ index 20) group. The results suggest that PPBP might be a novel promising serological marker and a prognostic factor specific to melanomas.     

Post-transplant proteinuria associated with everolimus: Definition of main features with proteomics

Little is known on both the composition and mechanism(s) of proteinuria associated with the use of mTOR inhibitors, in particular of Everolimus (E). We characterized urinary proteins utilizing an integrated proteomics approach (quantitative essays, 2‐DE, MALDI‐TOF, Western blot) in 48 renal transplant recipients who were alternatively treated with E (n = 31) or with enteric coated mycophenolic acid (EC‐MPA) (n = 17). Twelve E patients (39%) developed high (>3 g/day) or intermediate proteinuria (1–3 g) compared to four (23%) of the EC‐MPA group. Urinary proteins (p<0.001), β2 microglobulin (p<0.001) and α1microglobulin (p<0.025) were higher in E than in EC‐MPA, appeared more rapidly and were inversely correlated with the day of treatment. Proteomics showed a marked increase of all urinary components in E and EC‐MPA patients, major changes involving typical components of glomerular damage (albumin, α1‐Zn glycoprotein, α2HS glycoprotein, leucin‐richα2‐glycoprotein) and specific bio‐markers for E (clusters of α1‐antitrypsin fragments and monoclonal λ chains). Finally, inter‐α‐trypsin‐inhibitor heavy chain H4 precursor was decreased in E and EC‐MPA urine compared to normal urine. In conclusion, E induced massive and generalized proteinuria of mixed glomerular and tubular origin that was correlated with the start of treatment and reached a nephrotic range in few cases. Specific urinary markers reflect renal alterations related to the transplant or specific alterations associated with the drug.