Volume 47, Issue 6 - Special Issue: Proteomics and Biomarkers

Protein misfolding and deposition as amyloid, with consequent tissue damage, plays a key role in the group of diseases generically termed amyloidoses. In the systemic forms, amyloid deposition is widespread and causes severe dysfunction of vital organs. Proteomic analysis, thanks to its versatility and the comprehensiveness of information obtained, is an ideal tool for the study of systemic amyloidoses. It has been successfully employed in the characterization of the circulating amyloidogenic precursors and the analysis of affected tissues, for the diagnostic identification of the fibril components and for characterizing disease-related changes in protein expression. We present the developments in the field of proteomics applied to systemic amyloidoses, and discuss the perspectives opened in the study of these diseases. Clin Chem Lab Med 2009;47:627–35.

Proteomics technologies demonstrate enormous data-gathering capabilities to discover disease-specific biomarkers in serum, plasma, urine, tissue and other biological samples. The traditional way to achieve this consists of protein separation performed using two-dimensional polyacrylamide gel electrophoresis (2DE). However, the 2DE approach has its drawbacks with respect to automation, sensitivity, and throughput. Considerable efforts have been devoted to the development of non-gel-based proteome separation technologies able to resolve complex protein and peptide mixtures prior to mass spectrometric (MS) analysis. This review discusses some of the most recent advances in multidimensional peptide separation techniques compatible with MS analysis, including gel electrophoresis, isoelectric focusing, capillary electrophoresis and liquid chromatography techniques. Based on future perspectives and our experiences, special attention is given to the application of two-dimensional chromatographic separation coupled to MS, the so-called multidimensional protein identification technology approach. Clin Chem Lab Med 2009;47:636–46.

The importance of post-translational modifications (PTMs) of proteins has become evident in the proteomic era as it plays a critical role in modulating cellular function, and can vary in response to different stimuli thereby tuning cellular mechanisms. Assessment of PTMs on a proteomic scale is a challenging task since they are substoichiometric, transient and reversible. Moreover, the amount of post-translationally modified proteins is generally very small when compared to their unmodified counterparts. Existing methodologies for identification of PTMs essentially relies on enrichment procedure to selectively increase the amount of modified peptides. These procedures need to be integrated with sophisticated mass spectrometric methods to enable the identifications of PTMs. Although the strategies developed so far are not optimal, a number of examples will be given where the combination of innovative separation methods along with advanced mass spectrometric analyses provide positive results. These experiences are leading the way for the next generation of proteomic approaches for identification of a wide range of PTMs. Clin Chem Lab Med 2009;47:647–65.

Proteomics analysis has been heralded as a novel tool for identifying new and specific biomarkers that may improve diagnosis and monitoring of various disease states. Recent years have brought a number of proteomics profiling technologies. Although proteomics profiling has resulted in the detection of disease-associated differences and modification of proteins, current proteomics technologies display certain limitations that are hampering the introduction of these new technologies into clinical laboratory diagnostics and routine applications. In this review, we summarize current advances in mass spectrometry based biomarker discovery. The promises and challenges of this new technology are discussed with particular emphasis on diagnostic perspectives of mass-spectrometry based proteomics profiling for malignant diseases. Clin Chem Lab Med 2009;47:666–84.

Background : During clotting, α thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of α thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially α thrombin. Methods : Extrinsic addition of α thrombin to a subset (3–30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal α thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by α thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion. Results : Extrinsic addition of α thrombin to a subset (3–30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by α thrombin confirms these observations. Conclusions : The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum. Clin Chem Lab Med 2009;47:685–93.

Background : Issues have been raised concerning the robustness and validity of alleged serum markers discovered by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS). Pre-analytical variables have been shown to exert a profound effect on protein profiles, irrespective of true biological variation. However, little is known about the possible effects of sample storage duration on protein profiles. We, therefore, aimed to investigate the effects of extended storage duration on the serum protein profile. Methods : Archival sera from 140 breast cancer patients, stored at –30°C for 1–11 years, were profiled by SELDI-TOF MS using immobilised metal affinity capture (IMAC) arrays, a condition applied in the majority of breast cancer biomarker discovery studies. Results : Fourteen peak clusters, structurally identified as C3a des-arginine anaphylatoxin and multiple fragments of albumin and fibrinogen, were found to be significantly associated with sample storage duration by five distinct patterns. These proteins have been described previously as potential cancer markers, rendering them specific to both disease and sample handling issues. Conclusions : To prevent experimental variation being interpreted erroneously as disease associated variation, assessment of potential confounding pre-analytical parameters is a pre-requisite in biomarker discovery and validation studies. In addition, with respect to the different (non-)linear patterns observed in the current study, simply performing linear corrections to account for sample storage duration will not necessarily suffice. Clin Chem Lab Med 2009;47:694–705.

Background: The analysis of exhaled breath condensate (EBC) can be an alternative to traditional endoscopic sampling of lower respiratory tract secretions. This is a simple non-invasive method of diagnosing respiratory diseases, in particular, respiratory inflammatory processes. Methods: Samples were collected with a special device-condenser (ECoScreen, VIASYS Healthcare, Germany), then treated with trypsin according to the proteomics protocol for standard protein mixtures and analyzed by nanoflow high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Mascot software (Matrixscience) was used for screening the database NCBInr for proteins corresponding to the peptide maps that were obtained. Results: EBCs from 17 young healthy non-smoking donors were collected. Different methods for concentrating protein were compared in order to optimize EBC preparations for proteomic analysis. The procedure that was chosen allowed identification of proteins exhaled by healthy people. The major proteins in the condensates were cytoskeletal keratins. Another 12 proteins were identified in EBC from healthy non-smokers. Some keratins were found in the ambient air and may be considered exogenous components of exhaled air. Conclusions: Knowledge of the normal proteome of exhaled breath allows one to look for biomarkers of different disease states in EBC. Proteins in ambient air can be identified in the respiratory tract and should be excluded from the analysis of the proteome of EBC. The results obtained allowed us to choose the most effective procedure of sample preparation when working with samples containing very low protein concentrations. Clin Chem Lab Med 2009;47:706–12.

Background: Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF/MS), a laboratory-friendly technique, is used to identify biomarkers for cancer. The aim of the present study was to explore the application of SELDI proteomic patterns in serum for distinguishing between cases of pancreatic cancer, chronic pancreatitis, type 2 diabetes mellitus and healthy controls. Methods: Sera from 12 healthy controls, 24 patients with type 2 diabetes mellitus, 126 with pancreatic cancer, including 84 with diabetes, and 61 with chronic pancreatitis, 32 of which were diabetics, were analyzed using SELDI-TOF/MS. Spectra (IMAC-30) were clustered and classified using Biomarker Wizard and Biomarker Pattern software. Results: Two decision tree classification algorithms, one with and one without CA 19-9, were constructed. In the absence of CA 19-9, the splitting protein peaks were: m/z 1526, 1211, and 3519; when CA 19-9 was used in the analysis, it replaced the m/z 3519 splitter. The two algorithms performed equally for classifying patients. A classification tree that considered diabetic patients only was constructed; the main splitters were: 1211, CA 19-9, 7903, 3359, 1802. With this algorithm, 100% of patients with type 2 diabetes mellitus, 97% with chronic pancreatitis and 77% of patients with pancreatic cancer were correctly classified. SELDI-TOF/MS features improved the diagnostic accuracy of CA 19-9 (AUC=0.883 for CA 19-9; AUC=0.935 for CA 19-9 and SELDI-TOF/MS features combined). Conclusions: SELDI-TOF/MS allows identification of new peptides which, in addition to CA 19-9, enable the correct classification of the vast majority of patients with pancreatic cancer, which can be distinguished from patients with chronic pancreatitis or type 2 diabetes mellitus. Clin Chem Lab Med 2009;47:713–23.

The field of clinical proteomics offers opportunities to identify new disease biomarkers in body fluids, cells and tissues. These biomarkers can be used in clinical applications for diagnosis, stratification of patients for specific treatment, or therapy monitoring. New protein array formats and improved spectrometry technologies have brought these analyses to a level with potential for use in clinical diagnostics. The nature of the human body fluid proteome with its large dynamic range of protein concentrations presents problems with quantitation. The extreme complexity of the proteome in body fluids presents enormous challenges and requires the establishment of standard operating procedures for handling of specimens, increasing sensitivity for detection and bioinformatical tools for distribution of proteomic data into the public domain. From studies of in vitro diagnostics, especially in clinical chemistry, it is evident that most errors occur in the preanalytical phase and during implementation of the diagnostic strategy. This is also true for clinical proteomics, and especially for fluid proteomics because of the multiple pretreatment processes. These processes include depletion of high-abundance proteins from plasma or enrichment processes for urine where biological variation or differences in proteolytic activities in the sample along with preanalytical variables such as inter- and intra-assay variability will likely influence the results of proteomics studies. However, before proteomic analysis can be introduced at a broader level into the clinical setting, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement and data analysis needs to be improved. In this review, we discuss the recent technological advances and applications that fulfil the criteria for clinical proteomics, with the focus on fluid proteomics. These advances relate to preanalytical factors, analytical standardization and quality-control measures required for effective implementation into routine laboratory testing in order to generate clinically useful information. With new disease biomarker candidates, it will be crucial to design and perform clinical studies that can identify novel diagnostic strategies based on these techniques, and to validate their impact on clinical decision-making. Clin Chem Lab Med 2009;47:724–44.

Background : We investigated the genotypic distribution of Hp del in healthy subjects and cancer patients in Taiwan. Methods: Blood samples were collected from 244 randomly selected healthy Taiwanese volunteers and 737 patients with various cancers. Samples were analyzed for the haptoglobin (Hp) gene, and the presence of the Hp del allele was determined from genomic DNA by an Hp del -specific polymerase chain reaction (PCR) method. The plasma concentration of Hp was also determined. Results: The frequency of the Hp del allele was calculated to be 0.029, and was not different between the healthy subjects and patients with cancer. The prevalence of Hp deficiency caused by Hp del homozygosity was estimated to be ∼0.85 in 1000. Fifty-seven subjects were reclassified from homozygous Hp 1 or Hp 2 to Hp 1 /Hp del or Hp 2 /Hp del genotypes. The Hp del allele is not associated with prevalence, severity or stage of any cancer. Conclusions: Congenital Hp deficiency caused by Hp del homozygosity is a condition present in Taiwan with a relatively high frequency. However, the Hp del variant does not play a role in cancer. Clin Chem Lab Med 2009;47:745–9.

Background: The measurement of carbohydrate antigen 19-9 (CA 19-9) is recommended for the diagnosis and follow-up of pancreatic cancer. However, increased CA 19-9 has also been reported in patients with various benign diseases of the lung. We aimed to elucidate the pulmonary radiographic abnormalities and laboratory results associated with increased concentrations of CA 19-9. Methods: This study was performed using a case-controlled design. Cases included all participants in a cancer screening program who had an increased CA 19-9 concentration (>37 U/mL), but without a diagnosis of malignancy. Age- and sex-matched participants with normal CA 19-9 levels were enrolled as controls. Laboratory results and radiographic features were compared. Results: In total, 119 participants with increased CA 19-9 concentrations and 476 controls were included. A higher erythrocyte sedimentation rate (ESR) [adjusted odd ratio (aOR), 1.03; 95% confidence interval (CI), 1.01–1.05], higher hemoglobin A 1c (HbA 1c ) (aOR, 1.28; 95% CI, 1.05–1.56), bronchiectasis (aOR, 2.48; 95% CI, 1.22–5.02), bronchiolitis (aOR, 3.93; 95% CI, 1.88–8.22), emphysema (aOR, 2.67; 95% CI, 1.32–5.40), and interstitial fibrosis (aOR, 10.62; 95% CI, 2.03–55.44) were independent factors for increased CA 19-9. Conclusions: CA 19-9 concentrations, as well as increased ESR and HbA 1c , can be increased in patients with various lung abnormalities. Clin Chem Lab Med 2009;47:750–4.

Background: The differential diagnosis between bacterial and viral meningitis is not easy in some cases. Cerebrospinal fluid (CSF) analysis is essential for establishing this diagnosis. The objectives were to quantitate lactic acid (LA) concentrations in bacterial and viral meningitis, and other central nervous system (CNS) diseases in order to evaluate the diagnostic utility of CSF LA for discriminating bacterial from viral meningitis. Methods: CSF LA was measured in 139 CSF samples from seven groups: viral meningitis with classic CSF; suspicion of viral meningitis with neutrophils in CSF; bacterial meningitis; non-infectious neurological diseases; chronic meningitis; traumatic lumbar puncture (LP) and normal CSF. Results: CSF LA was higher in bacterial meningitis 8.7+5.4 mmol/L compared with viral meningitis (1.9+0.6) and the other groups (p<0.0001). CSF LA in the groups with viral meningitis was not different compared to groups with non-infectious CNS diseases and chronic meningitis. The ability of CSF LA to discriminate bacterial from viral meningitis showed a sensitivity of 80% and specificity of 97%, positive predictive value (PPV) of 94%, and negative predictive value (NPV) of 89%. Conclusions: CSF LA is a powerful test to discriminate bacterial from viral meningitis with high sensitivity, specificity and predictive values. CSF LA can help in the cases with diagnostic uncertainty. Clin Chem Lab Med 2009;47:755–61.

Background : We organized and conducted a proficiency testing study (CardioOrmocheck) to evaluate the differences in analytical performance of brain natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) immunoassays. Methods : Approximately 90 Italian laboratories were involved in the 2005–2007 proficiency testing cycles, while 112 laboratories took part in the 2008 cycle (from January to May 2008). A total of 28 study samples were measured by participating laboratories for a total of 2354 determinations. Results : The mean total variability for BNP (50.6 %CV) was significantly higher than that for NT-proBNP (8.4 %CV). In addition, the mean variability due to differences between-methods (46.4 %CV) comprised the majority of the total variability for BNP. Between-method variability for BNP comprised, on average, 84% of total variability, while the within-method variability comprised an average of 20.2 %CV. On the contrary, for NT-proBNP the within-method variability (7.3 %CV) represented the majority of total variability (average 75%), while between-method variability was smaller (4.1 %CV). Imprecision around the cut-off value showed marked differences among methods, especially for BNP immunoassay methods. In addition, BNP methods were affected by large systematic differences, for example an average 2.7-fold difference between Access and ADVIA Centaur methods, while agreement between NT-proBNP methods was better (an average 1.2-fold difference between Dimension and ECLIA on the Elecsys methods). Conclusions : This multicenter collaborative study demonstrates that there are significant differences in analytical characteristics and measured values among the most popular commercial methods for BNP and NT-proBNP. Clinicians should be very careful when comparing results obtained by laboratories that use different methods. Clin Chem Lab Med 2009;47:762–8.

Background: Venous blood sampling is usually performed using a tourniquet to help locate and define peripheral veins to achieve successful and safe venipuncture. Despite widespread usage of tourniquets for venipuncture by medical and laboratory staff, very few are aware of the effects of tourniquet application on laboratory parameters. In addition, definitive guidelines regarding when and how to use a tourniquet for blood sampling are lacking. The aim of the present study was to define the optimal sampling time after tourniquet removal to avoid adverse impact on laboratory analytes. Methods: Blood oxygen and carbon dioxide partial pressure, pH, oxyhemoglobin saturation (satO 2 ), hematological parameters, serum electrolyte concentrations, erythrocyte, deformability and aggregation, leukocyte activation and nitrite/nitrate concentrations obtained 180 s after tourniquet release were compared with baseline values for 10 healthy subjects. Results: Blood gases, hematological parameters and serum electrolyte levels were not affected by the application and removal of a tourniquet. However, there were significant decreases in erythrocyte deformability at 90, 120, 180 s, and increases in erythrocyte aggregation at 5 and 30 s following removal of the tourniquet. A significant increase in granulocyte respiratory burst at 60 s was observed, confirming leukocyte activation due to application of the tourniquet. There were no significant alterations of blood nitrite/nitrate levels. Conclusions: Our blood sampling technique which mimicked the application and release of a tourniquet indicated unaltered values for routine blood gases, hematological testing and serum electrolyte levels. Conversely, hemorheological measurements can be affected. Therefore, it is strongly recommended that tourniquet application should be avoided during blood sampling or, if this is not possible, the procedure should be well standardized and details of the sampling method should be reported. Clin Chem Lab Med 2009;47:769–76.

Background: Muscle adaptation which occurs following eccentric exercise-induced muscle damage has been associated with an acute inflammatory response. The purpose of this study was to investigate serum interleukin-6 (IL-6), osteoprotegerin and receptor activator of nuclear factor kB ligand (OPG/RANKL) concentrations following muscle damage. We measured changes for several days following muscle damage. Methods: Ten healthy young males performed an eccentric exercise protocol using their quadriceps. Blood samples were withdrawn before and at 6 h, 2 days, 5 days and 16 days post-exercise. Functional and clinical measurements were performed before, and on days 1, 2, 5, 8, 12 and 16 post-exercise. Results: The exercise protocol resulted in muscle damage, indicated by changes in biochemical markers. An increase in IL-6 and OPG, and a decrease in RANKL concentrations were seen at 6 h and on day 2 post-exercise; the OPG:RANKL ratio was increased at 6 h post-exercise (p<0.05). Conclusions: Changes in IL-6 and OPG/RANKL system may represent systemic responses in muscle inflammation and repair processes. However, further studies are needed to elucidate a potential systemic and/or local role of the OPG/RANKL system in skeletal muscle repair. Clin Chem Lab Med 2009;47:777–82.