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.

     

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.     

Towards stable diagnostic setups in clinical proteomics: Absolute quantitation of peptide biomarkers using MALDI‐TOF‐MS

In routine clinical diagnostics, peptide biomarkers are most commonly quantified using immunological techniques but these methods often lack sensitivity and/or specificity. Hence, quantitative mass spectrometry detection is desirable as an alternative diagnostic tool. To date, quantitative mass spectrometry is mostly based on ESI‐MS coupled to LC, requiring highly sophisticated instrumentation and knowledge and is time consuming and expensive. In contrast, MALDI‐TOF‐MS is a very simple, sensitive and rapid method for the detection of peptide biomarkers. However, the infeasibility of absolute quantification has been a tremendous handicap to the use of MS in stable clinical diagnostics. Here, we describe the development of a technical platform based on ClinProt particles and heavy‐isotope internal peptide standards for the fast and reliable preparation of samples. This combines the advantages of MALDI‐TOF as a read‐out system with absolute quantitation of peptide biomarkers. As a proof‐of‐concept, this platform was successfully employed for the absolute determination of the concentration of the highly abundant serum peptide des‐Ala‐Fibrinopeptide A in 45 serum samples from healthy donors. Such technology essentially contributes to the development of a stable MALDI‐TOF‐MS‐based clinical assay.     

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.     

Towards defining the whole salivary peptidome

We report the identification of 2294 peptides/proteins in whole saliva in the mass range between 700 and 16 000 Da by LC‐MS and MS/MS using a MALDI‐TOF/TOF mass spectrometer. Most of the identified peptides/proteins are originated from cellular debris or plasma components while only 26% (n = 607) correspond to salivary peptides/proteins species. In spite of the presence of the major salivary peptides in all samples from the six subjects analyzed, each individual presents a different pattern of fragments, many deriving from the same protein sequence. All our data, in particular the large number of fragments found, suggest high proteolytic activity insight the oral cavity. The analysis of samples by gelatin zymography showed that all saliva donors displayed multiple proteolytic bands, two identified as cathepsin D and G by MS. Analysis of the cleavage site distribution on the main peptide sequences based on contingency tables shows that the predominant cleavages occur between Gln‐Gly or Tyr‐Gly. These cleavages are largely associated with proline‐rich proteins peptides and with histatin 1 and P‐B peptide, respectively. However, depending on the peptide class, different cleavage hits were observed suggesting the presence of a set of proteases acting in different ways according to different peptide sequences. Comparing the number of cleavages involving all residues, it is possible to observe that 44% (±10%) of the observed cleavages in histatin, statherin and P‐B peptide in all individuals may be explained by cathepsin D, suggesting a major role for this enzyme in oral cavity proteolysis.     

Endogeneous peptide profiling of cerebrospinal fluid by MALDI-TOF mass spectrometry: Optimization of magnetic bead-based peptide capture and analysis of preanalytical variables

Cerebrospinal fluid (CSF) perfuses the brain and spinal cord. CSF contains peptides and proteins important for brain physiology and potentially also relevant to brain pathology. High-throughput endogeneous peptide profiling by MS is an emerging approach for disease diagnosis and biomarker discovery. A magnetic bead-based method for off-line serum peptide capture coupled to MALDI-TOF-MS has been introduced recently. In this study, we optimize the peptide capture method for profiling of CSF and investigate the effect of a number of preanalytical variables. The CSF profiles contain ～100 reliably detected peptides at m/z 800-4000 with reproducible ion intensities (average 7% CV). The investigated preanalytical variables include: time at room temperature (RT) before storage, storage temperature, freeze-thawing cycles, and blood contamination. The CSF peptidome (<20?kDa) is relatively stable and can withstand a few hours at RT and several freeze-thaw cycles. Several peptides sensitive to storage at -20°C, including Cystatin C, were assigned based on mass or identified by MS/MS. Hemoglobin α and β chains were detected in blood contaminated samples, at levels invisible to the eye (0.01%). These peptides may be used for quality control in a MALDI-TOF-MS screening strategy to select high quality samples for in-depth proteomics analysis in disease studies.     

Thymosin β4 is differentially expressed in the cerebrospinal fluid of Creutzfeldt‐Jakob disease patients: a MALDI‐TOF MS protein profiling study

MALDI‐TOF protein profiling analysis permits the detection of peptides and small proteins in complex protein mixtures with great accuracy. We applied this analysis to cerebrospinal fluid (CSF) from 15 patients affected by Creutzfeldt‐Jakob disease (CJD). We compared the levels of the normalized ion signals of 11 sporadic and 4 genetic CJD forms with those from ten healthy control subjects and eight non‐CJD relapsing‐remitting multiple sclerosis patients. In so doing, we detected 61 differentially expressed ion signals in CJD samples compared to controls. Among the 61 signals, 3 signals had significantly increased levels with high statistical significance (p <0.0001) and were located at 3238.3 m/z, 4963.7 m/z, and 8565.3 m/z. We characterized the 5.0 and 8.6 kDa proteins as thymosin β4 N‐acetylated and free ubiquitin, respectively, while the 3.2‐kDa peptide remained uncharacterized. Although elevated ubiquitin levels have previously been described in CJD, we have demonstrated for the first time the involvement of thymosin β4 in a neurodegenerative disease. To support the validity of thymosin β4 levels obtained by MALDI‐TOF analysis, an independent enzyme immunoassay analysis was performed. Moreover, a validation cohort consisting of CSF from three CJD patients, five healthy subjects, and six non‐CJD relapsing‐remitting multiple sclerosis patients was analyzed in a similar way, yielding superimposable results. We propose that thymosin β4 is a potential new candidate marker for the ante mortem diagnosis of CJD disease.     

Salivary biomarkers associated with perceived satiety and body mass in humans

Regulation of food intake and energy homeostasis is controlled by a delicate balancing of numerous central and peripheral factors, including circulating peptide hormones. This study investigated the proteome of saliva using SELDI‐TOF‐MS in relation to satiety and body mass index (BMI) in humans. Within a longitudinal test session, 18 subjects were exposed to a lunch‐induced hunger‐satiety shift, while every 15 min collecting their whole saliva and rating their hunger and satiety. Saliva was analysed by SELDI‐TOF‐MS using IMAC arrays with a chromatographic copper surface (IMAC‐Cu). From all subjects and time points measured, peptide and protein profiles showed 190 common peaks. Their interrelationships show that 37% of the variation was accounted for in one dimension. About 30 means had a strong association (0.70<|r|<0.95) with all subjective satiety ratings across time during the test session, and seven peaks were significantly correlated to BMI. Database MS searches indicated characterisation of some relevant metabolic peptide hormones. In conclusion, SELDI‐TOF‐MS on human saliva provides a valuable and noninvasive way of profiling that enables characterisation of novel and differently expressed peptides and proteins which can be used as biomarkers of satiety and overweight.     

Two dimensional gel electrophoresis of apolipoprotein C‐III and MALDI‐TOF MS are complementary techniques for the study of combined defects in N‐ and mucin type O‐glycan biosynthesis

In the field of diseases related to glycosylation disorders, congenital defects associated with abnormalities in both O‐ and N‐glycosylation of proteins constitute arising novel entities. Defects in subunits of the conserved oligomeric Golgi protein complex have been shown to be involved in an important part of previously unsolved CDG type II combining abnormalities in both mucin type core1 O‐ and N‐glycans; furthermore, recent studies revealed that autosomal recessive cutis laxa type II could also be associated with such combined glycosylation defects. Based on the studies of serum samples from three patients including a case of cutis laxa, we present here evidence that 2‐DE of apolipoprotein C‐III in combination with MALDI‐TOF‐MS analysis of serum O‐ and N‐glycans allow the detection and the biochemical characterization of these newly recognized glycosylation disorders.     

Identification of distinctive protein expression patterns in colorectal adenoma

Purpose: As a pre‐malignant precursor, adenoma provides an ideal tissue for proteome profiling to investigate early colorectal cancer development and provide possible targets for preventive interventions. The aim of this study was to identify patterns of differential protein expression that distinguish colorectal adenoma from normal tissue. Experimental design: Twenty paired samples of adenoma and normal mucosa were analysed by 2‐DE and MALDI‐TOF/TOF MS to detect proteins with ≥2‐fold differential expression. Results: Four proteins were up‐regulated in adenoma (Annexin A3, S100A11, S100P and eIF5A‐1) and three were down‐regulated (Galectin‐1, S100A9 and FABPL). S100P, galectin‐1, S100A9 and FABPL expression was localised by immunohistochemistry. Conclusions and clinical relevance: Distinctive patterns of in vivo protein expression in colorectal adenoma were identified for the first time. These proteins have important functions in cell differentiation, proliferation and metabolism, and may play a crucial role in early colorectal carcinogenesis. The ability to recognise premalignant lesions may have important applications in cancer prevention.     

The feasibility of MS and advanced data processing for monitoring Schistosoma mansoni infection

Purpose : Sensitive diagnosis, monitoring of disease progression and the evaluation of chemotherapeutic interventions are of prime importance for the improvement of control and prevention strategies for Schistosomiasis. The aim of the present study was to identify novel markers of Schistosoma mansoni infection and disease using urine samples from a large cohort from an area endemic for S. mansoni. Experimental design : Urine samples were collected and processed on an automated sample clean‐up and fractionation system combining strong cation exchange and reversed phase, and analyzed by MS (MALDI ToF MS). The ClinPro Tools^? (CPT) software and the Discrete Wavelet Transformation–Support Vector Machine (DWT‐SVM) procedure were used for classification and statistical analysis. Results : We observed a large difference in urinary peptide profiles between children and adults but classification based on infection was possible only for children. Here, in the external validation data set, 93% of the infected children were classified correctly with DWT‐SVM (versus 76% for CPT). In addition 91% of low‐infected children were classified correctly using DWT‐SVM (versus 85% for CPT). The discriminating peptides were identified as fragments of collagen 1A1 and 1A3, and uromodulin. Conclusions and clinical relevance : In conclusion, we provide the usefulness of a peptidomics profiling approach combined with DWT‐SVM in the monitoring of S. mansoni infection.     

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre‐clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI‐TOF‐MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre‐albumin) and C3a‐desArg by MS/MS and were further validated by antibody‐based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut‐off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a‐desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.     

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 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.     

Changes of serum‐associated fucosylated glycoproteins and changes in glycosylation of IgA in human cirrhosis

Many modifications in N‐glycosylation have been demonstrated in hepatic cirrhosis. These modifications correspond to an increase of a bisecting core alpha(1,6)‐fucosylated biantennary glycan, an increase in core fucosylation, and the presence of an important population of neutral oligosaccharides in human serum of cirrhotic patients. In this study, a glycoproteomic approach which consists of lectin affinity chromatography, MALDI‐TOF MS for the characterization of N‐glycans released from glycoproteins, one‐ and 2‐D PAGE, electrospray ionization quadrupole ion trap (ESI‐QIT) MS was used to identify serum fucosylated glycoproteins related to cirrhosis. Employing this method, we have shown that IgA is one of the major proteins that is responsible of the glycosylation modifications observed in the serum N‐glycome of cirrhotic patients. To our knowledge, this is the first time that aberrant N‐glycosylation of IgA in cirrhosis is described.     

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.     

Protein profiling in pathology: analysis and evaluation of 239 frozen tissue biopsies for diagnosis of B-cell lymphomas

Purpose : We determined the potential value of protein profiling of tissue samples by assessing how precise this approach enables discrimination of B‐cell lymphoma from reactive lymph nodes, and how well the profiles can be used for lymphoma classification. Experimental design : Protein lysates from lymph nodes (n=239) from patients with the diagnosis of reactive hyperplasia (n=44), follicular lymphoma (n=63), diffuse large B‐cell lymphoma (n=43), mantle cell lymphoma (n=47), and chronic lymphocytic leukemia/small lymphocytic B‐cell lymphoma (n=42) were analysed by SELDI‐TOF MS. Data analysis was performed by (i) classification and regression tree‐based analysis and (ii) binary and polytomous logistic regression analysis. Results : After internal validation by the leave‐one‐out principle, both the classification and regression tree and logistic regression classification correctly identified the majority of the malignant (87 and 96%, respectively) and benign cases (73 and 75%, respectively). Classification was less successful since approximately one‐third of the cases of each group were misclassified according to the histological classification. However, an additional mantle cell lymphoma case that was misclassified as chronic lymphocytic leukemia/small lymphocytic B‐cell lymphoma initially was identified based on the protein profile. Conclusions and clinical relevance : SELDI‐TOF MS protein profiling allows for reliable identification of the majority of malignant lymphoma cases; however, further validation and testing robustness in a diagnostic setting is needed.     

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.