Systematic quantification of peptides/proteins in urine using selected reaction monitoring

The evaluation of biomarkers in bodily fluids necessitates the development of robust methods to quantify proteins in a complex background, using large sets of samples. The ability to multiplex numerous analytes in a single assay expedites the process. Liquid chromatography-mass spectrometry (LC-MS) analyses performed in selected reaction monitoring (SRM) in conjunction with stable isotope dilution MS present an effective way to detect and quantify biomarker candidates in bodily fluids. The strategy presented involves an initial qualification of predefined sets of proteins in urine. The technique was applied to detect and quantify peptides in urine samples as surrogates for a few endogenous proteins. Multiplexed assays were developed to analyze proteins associated with bladder cancer; a few exogenous proteins were added as internal standards. The sample preparation and the analytical protocols were optimized to ensure reproducibility, analytical precision, and quantification limits in the low nanogram per milliliter range. Analyses were performed using known amounts of isotopically labeled peptides. Systematic replication of the measurements indicated intra-assay and inter-assay variability, with CVs in the range of 10%. The differences measured for two targeted proteins were correlated with their level of expression in the corresponding tumors using immunohistochemistry.     

Assessment of Oxidative Damage to Proteins and DNA in Urine of Newborn Infants by a Validated UPLC-MS/MS Approach

The assessment of oxidative stress is highly relevant in clinical Perinatology as it is associated to adverse outcomes in newborn infants. This study summarizes results from the validation of an Ultra Performance Liquid Chromatography–tandem Mass Spectrometry (UPLC-MS/MS) method for the simultaneous quantification of the urinary concentrations of a set of endogenous biomarkers, capable to provide a valid snapshot of the oxidative stress status applicable in human clinical trials, especially in the field of Perinatology. The set of analytes included are phenylalanine (Phe), para-tyrosine (p-Tyr), ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-NO₂-tyrosine (3NO₂-Tyr), 3-Cl-tyrosine (3Cl-Tyr), 2′-deoxyguanosine (2dG) and 8-hydroxy-2′-deoxyguanosine (8OHdG). Following the FDA-based guidelines, appropriate levels of accuracy and precision, as well as adequate levels of sensitivity with limits of detection (LODs) in the low nanomolar (nmol/L) range were confirmed after method validation. The validity of the proposed UPLC-MS/MS method was assessed by analysing urine samples from a clinical trial in extremely low birth weight (ELBW) infants randomized to be resuscitated with two different initial inspiratory fractions of oxygen.     

Antibody‐based proteomics and biomarker research—Current status and limitations

Antibody‐based proteomics play a very important role in biomarker discovery and validation, facilitating the high‐throughput evaluation of candidate markers. Most proteomics‐driven discovery is nowadays based on the use of MS. MS has many advantages, including its suitability for hypothesis‐free biomarker discovery, since information on protein content of a sample is not required prior to analysis. However, MS presents one main caveat which is the limited sensitivity in complex samples, especially for body fluids, where protein expression covers a huge dynamic range. Antibody‐based technologies remain the main solution to address this challenge since they reach higher sensitivity. In this article, we review the benefits and limitations of antibody‐based proteomics in preclinical and clinical biomarker research for discovery and validation in body fluids and tissue. The combination of antibodies and MS, utilizing the best of both worlds, opens new avenues in biomarker research.     

Mass spectrometry-based expression profiling of clinical prostate cancer

The maturation of MS technologies has provided a rich opportunity to interrogate protein expression patterns in normal and disease states by applying expression protein profiling methods. Major goals of this research strategy include the identification of protein biomarkers that demarcate normal and disease populations, and the identification of therapeutic biomarkers for the treatment of diseases such as cancer (Celis, J. E., and Gromov, P. (2003) Proteomics in translational cancer research: Toward an integrated approach. Cancer Cell 3, 9-151). Prostate cancer is one disease that would greatly benefit from implementing MS-based expression profiling methods because of the need to stratify the disease based on molecular markers. In this review, we will summarize the current MS-based methods to identify and validate biomarkers in human prostate cancer. Lastly, we propose a reverse proteomic approach implementing a quantitative MS research strategy to identify and quantify biomarkers implicated in prostate cancer development. With this approach, the absolute levels of prostate cancer biomarkers will be identified and quantified in normal and diseased samples by measuring the levels of native peptide biomarkers in relation to a chemically identical but isotopically labeled reference peptide. Ultimately, a centralized prostate cancer peptide biomarker expression database could function as a repository for the identification, quantification, and validation of protein biomarker(s) during prostate cancer progression in men.     

DeepQuanTR: MALDI‐MS‐based label‐free quantification of proteins in complex biological samples

The quantification of changes in protein abundance in complex biological specimens is essential for proteomic studies in basic and applied research. Here we report on the development and validation of the DeepQuanTR software for identification and quantification of differentially expressed proteins using LC‐MALDI‐MS. Following enzymatic digestion, HPLC peptide separation and normalization of MALDI‐MS signal intensities to the ones of internal standards, the software extracts peptide features, adjusts differences in HPLC retention times and performs a relative quantification of features. The annotation of multiple peptides to the corresponding parent protein allows the definition of a Protein Quant Value, which is related to protein abundance and which allows inter‐sample comparisons. The performance of DeepQuanTR was evaluated by analyzing 24 samples deriving from human serum spiked with different amounts of four proteins and eight complex samples of vascular proteins, derived from surgically resected human kidneys with cancer following ex vivo perfusion with a reactive ester biotin derivative. The identification and experimental validation of proteins, which were differentially regulated in cancerous lesions as compared with normal kidney, was used to demonstrate the power of DeepQuanTR. This software, which can easily be used with established proteomic methodologies, facilitates the relative quantification of proteins derived from a wide variety of different samples.     

Selected reaction monitoring for quantitative proteomics: a tutorial

Systems biology relies on data sets in which the same group of proteins is consistently identified and precisely quantified across multiple samples, a requirement that is only partially achieved by current proteomics approaches. Selected reaction monitoring (SRM)—also called multiple reaction monitoring—is emerging as a technology that ideally complements the discovery capabilities of shotgun strategies by its unique potential for reliable quantification of analytes of low abundance in complex mixtures. In an SRM experiment, a predefined precursor ion and one of its fragments are selected by the two mass filters of a triple quadrupole instrument and monitored over time for precise quantification. A series of transitions (precursor/fragment ion pairs) in combination with the retention time of the targeted peptide can constitute a definitive assay. Typically, a large number of peptides are quantified during a single LC‐MS experiment. This tutorial explains the application of SRM for quantitative proteomics, including the selection of proteotypic peptides and the optimization and validation of transitions. Furthermore, normalization and various factors affecting sensitivity and accuracy are discussed.     

Use of narrow‐range peptide IEF to improve detection of lung adenocarcinoma markers in plasma and pleural effusion

In this study we applied narrow‐range peptide IEF to plasma or pleural effusion prior to LC/MS/MS. Two methods for narrow‐range IEF were run; IPG strips and free‐flow electrophoresis. Data from this study was compared with cell line data to evaluate the method performance in body fluids. To test the methods potential in quantitative biomarker discovery studies, plasma and pleural effusion from patients with lung adenocarcinoma (n=3) were compared with inflammatory pleuritis (n=3) using iTRAQ quantification. Using narrow‐range IEF on the peptide level we were able to identify and quantify 282 proteins in plasma and 300 proteins in pleural effusion. These body fluid proteomes demonstrated high degree of overlap; however, more proteins significantly differently altered levels related to adenenocarcinoma were found in pleural effusion compared with plasma, suggesting enrichment of lung tissue‐related proteins in pleural effusion. Nine proteins were chosen for initial validation with Western blot, and one protein (NPC2) was chosen for further validation using imunohistochemistry. Overall, the quantitative results from IEF/LC/MS/MS showed good correlation with the results from Western blot and imunohistochemistry, showing the potential of this methodology in quantitative biomarker discovery studies.     

The utility of stable isotope labeled (SIL) analogues in the bioanalysis of endogenous compounds by LC-MS applied to the study of bile acids in a metabolomics assay

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC–MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC–MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.     

A simple and robust UPLC-SRM/MS method to quantify urinary eicosanoids

Eicosanoids are key mediators and regulators of inflammation and oxidative stress often used as biomarkers for diseases and pathological conditions such as cardiovascular and pulmonary diseases and cancer. Analytically, comprehensive and robust quantification of different eicosanoid species in a multi-method approach is problematic because most of these compounds are relatively unstable and may differ in their chemical properties. Here we describe a novel ultra-performance liquid chromatography-selected reaction monitoring mass spectroscopy (UPLC-SRM/MS) method for simultaneous quantification of key urinary eicosanoids, including the prostaglandins (PG) tetranor PGE-M, 8-iso-, and 2,3-dinor-8-iso-PGF_2α; the thromboxanes (TXs) 11-dehydro- and 2,3-dinor-TXB₂; leukotriene E₄; and 12-hydroxyeicosatetraenoic acid. In contrast to previous methods, which used time-consuming and complex solid phase extraction, we prepared samples with a simple liquid/liquid extraction procedure. Because collision-induced dissociation produced characteristic product ions for all analytes, no derivatization step for SRM/MS analysis was necessary. Analytes were separated with a short UPLC reversed-phase column (1.7 µm particles), allowing shorter run times than conventional HPLC columns. The method was validated and applied to human urine samples showing excellent precision, accuracy, detection limits, and robustness. In summary, the developed method allows robust and sensitive profiling of urinary eicosanoid species, making it a useful and valuable tool for biomarker profiling in clinical/toxicological studies.     

Validation of a dual LC–HRMS platform for clinical metabolic diagnosis in serum,bridging quantitative analysis and untargeted metabolomics

Mass spectrometry-based metabolomics is a rapidly growing field in both research and diagnosis. Generally, the methodologies and types of instruments used for clinical and other absolute quantification experiments are different from those used for biomarkers discovery and untargeted analysis, as the former requires optimal sensitivity and dynamic range, while the latter requires high resolution and high mass accuracy. We used a Q-TOF mass spectrometer with two different types of pentafluorophenyl (PFP) stationary phases, employing both positive and negative ionization, to develop and validate a hybrid quantification and discovery platform using LC–HRMS. This dual-PFP LC–MS platform quantifies over 50 clinically relevant metabolites in serum (using both MS and MS/MS acquisitions) while simultaneously collecting high resolution and high mass accuracy full scans to monitor all other co-eluting non-targeted analytes. We demonstrate that the linearity, accuracy, and precision results for the quantification of a number of metabolites, including amino acids, organic acids, acylcarnitines and purines/pyrimidines, meets or exceeds normal bioanalytical standards over their respective physiological ranges. The chromatography resolved highly polar as well as hydrophobic analytes under reverse-phase conditions, enabling analysis of a wide range of chemicals, necessary for untargeted metabolomics experiments. Though previous LC–HRMS methods have demonstrated quantification capabilities for various drug and small molecule compounds, the present study provides an HRMS quant/qual platform tailored to metabolic disease; and covers a multitude of different metabolites including compounds normally quantified by a combination of separate instrumentation.     

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.     

Urinary metanephrines by liquid chromatography tandem mass spectrometry: using multiple quantification methods to minimize interferences in a high throughput method

Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC-MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L. The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC-MS method. Injection-to-injection time was 6 min. The validated LC-MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well.     

Application of the standard addition approach for the quantification of urinary benzene

Urinary benzene is used as biomarker of exposure to evaluate the uptake of this solvent both in non-occupationally exposed population and in benzene-exposed workers. The quantitative determination of benzene in urine is carried out in a three steps procedure: urine collection, sample analysis by head space/solid phase microextraction/gas chromatography/mass spectrometry and analyte quantification. The adopted quantification method influences the initial step, hence the whole procedure. Two quantification approaches were compared as regards precision and accuracy: the calibration curves and the standard addition method. Even if calibration curves obtained by using urine samples from different subjects were always linear, their slopes and intercepts showed noteworthy variations, attributable to the influence of the biological matrix on benzene recovery. The standard addition method showed to be more suitable for compensating matrix effects, and a three-point standard addition protocol was used to quantify benzene in urine samples of 11 benzene-exposed workers (smokers and non-smokers). Urine from occupationally exposed workers was collected before and after work-shift. Besides urinary benzene, the applicability of the method was verified by measuring the urinary concentration of the S-phenylmercapturic acid, a specific benzene metabolite, generally adopted as biomarker in biological monitoring procedures. A similar trend of concentration levels of both analytes measured in urine samples collected before work-shift with respect to the after work-shift ones was found, showing the actual applicability of the standard addition method for biological monitoring purposes.     

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LCMSMS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3–8.7%) and accuracy (93–105%) across the calibration range of 0.1 to 50 ng/ml using an 80 μl plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LCMSMS is applicable for the quantification of other pharmaceuticals in pharmacokinetics studies in animals and humans which require high sensitivity.     

Bioanalysis in clinical development of tasquinimod using liquid chromatography/tandem mass spectrometry

Tasquinimod (ABR-215050) is an oral drug in clinical development for treatment of patients with castrate resistant prostate cancer. This paper describes a method for the determination of tasquinimod in human plasma. The method is based on liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) using stable isotope labeled tasquinimod as internal standard (IS). The plasma samples were processed by protein precipitation using acidic acetonitrile containing the IS. The precipitated samples were centrifuged and the supernatant was injected directly into the LC-MS/MS system. Chromatographic separation was performed on a reversed phase column using fast gradient elution, with a total run cycle time of 4 min. The method was validated with respect to accuracy, precision, dynamic range, lower limit of quantification, selectivity and robustness. Furthermore, the stability of tasquinimod in spiked plasma, in processed extracts and in incurred samples was thoroughly studied. The method was validated in the range of 1.0-2400 nmol/L, defining the lower and upper limits of quantification. The repeatability, reproducibility and overall bias were 1.5-7.1%, 3.5-7.4%, and 1.3-4.7%, respectively, in the range of 1-2000 nmol/L. Excellent selectivity was demonstrated in the validation, as well as in study samples from both healthy volunteers and cancer patients. Robustness was demonstrated by the calculated carry-over as low as 0.06%, and by an incurred sample reproducibility (ISR) experiment where 97% of the reanalyzed samples fulfilled the acceptance criteria of 20% deviation from initial analysis result. Also, tasquinimod was found to be stable in all investigated matrices, including in incurred samples. In an incurred sample stability (ISS) investigation, tasquinimod was demonstrated to be stable for 24 months, and 97% of the reanalyzed samples were within 20% from the initial analysis result. In conclusion, the method was demonstrated to be accurate, precise, robust and reliable for the determination of tasquinimod. The method was successfully used in several clinical studies for the support of pharmacokinetic and pharmacodynamic evaluations.     

Simultaneous determination of reduced glutathione,glutathione disulphide and glutathione sulphonamide in cells and physiological fluids by isotope dilution liquid chromatography–tandem mass spectrometry

A stable isotope dilution liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneously quantifying glutathione (GSH), glutathione disulphide (GSSG) and glutathione sulphonamide (GSA) from biological samples. GSA is a selective product of the reaction of GSH with hypochlorous acid and a potential biomarker of myeloperoxidase activity. GSH was detected as the N-ethylmaleimide alkylated adduct, as formation of this species prevented GSH oxidation occurring during sample processing. Synthesised stable isotope analogues were used as internal standards to accurately quantify each target species. The limit of quantification was determined as being 0.1 pmol for each species and excellent linearity was observed over relevant concentration ranges for biological samples. Relative standard deviations were <5% for within-day variation and <10% for between-day variation, except at the lower limit of quantification where they remained <20%. Accuracy was between 82% and 113%. We could detect GSA in neutrophils and endothelial cells treated with hypochlorous acid and in bronchoalveolar lavage fluid from children with cystic fibrosis. This is the first time GSA has been quantified in clinical material and suggests it is formed in vivo. The assay can now be used for investigating GSA as a biomarker of myeloperoxidase activity in inflammatory conditions, and is also applicable to measuring GSH:GSSG molar ratios as a general index of oxidative stress.     

A comprehensive peptidome profiling technology for the identification of early detection biomarkers for lung adenocarcinoma

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens.     

Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma.

Two mass spectrometric methods were established for the quantitative analyses of alpha-tocopherol (TH) and its oxidation product alpha-tocopherolquinone (TQ) in human plasma. Both methods make use of isotopically labeled internal standards of different levels of deuteration (d3-TH and d6-TQ). Plasma (100 microl) was saponified in the presence of a mixture of antioxidants, and then TH and TQ were extracted with hexane. With the GC-MS method, the analytes were first converted into O-trimethylsilyl derivatives before analysis in the selective ion monitoring mode. The derivatization procedure led to the quantitative conversion of TQ into the O-trimethylsilyl derivative of tocopherolhydroquinone, giving rise to a more stable molecule with less fragmentation than for TQ. The increased stability of the molecule resulted in an enhanced contribution of the base peak to the total observed ions and therefore an increased sensitivity of the base peak for quantification. With the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, TH and TQ were detected by multiple reaction monitoring after positive electrospray ionization. The GC-MS and LC-MS/MS methods showed nearly the same accuracy (>95%) and the same within-day precisions, with less than 5 and 10% for TH and TQ, respectively. The between-day precision and the limit of quantification for TQ in plasma were better by LC-MS/MS (4%; 3 nM) than by GC-MS (21%; 10 nM). Analysis and method validation were carried out with plasma samples obtained from a male volunteer pre- and postexercise. Both techniques showed that the ratio of TQ/TH was elevated by 35% immediately after exercise and had returned to basal levels when measured 24 h later.     

The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

Mass spectrometry (MS) -based proteomics has become an indispensable tool with broad applications in systems biology and biomedical research. With recent advances in liquid chromatography (LC) and MS instrumentation, LC–MS is making increasingly significant contributions to clinical applications, especially in the area of cancer biomarker discovery and verification. To overcome challenges associated with analyses of clinical samples (for example, a wide dynamic range of protein concentrations in bodily fluids and the need to perform high throughput and accurate quantification of candidate biomarker proteins), significant efforts have been devoted to improve the overall performance of LC–MS-based clinical proteomics platforms. Reviewed here are the recent advances in LC–MS and its applications in cancer biomarker discovery and quantification, along with the potentials, limitations and future perspectives.     

Proteomic data analysis workflow for discovery of candidate biomarker peaks predictive of clinical outcome for patients with acute myeloid leukemia

Our goal in this paper is to show an analytical workflow for selecting protein biomarker candidates from SELDI-MS data. The clinical question at issue is to enable prediction of the complete remission (CR) duration for acute myeloid leukemia (AML) patients. This would facilitate disease prognosis and make individual therapy possible. SELDI-mass spectrometry proteomics analyses were performed on blast cell samples collected from AML patients pre-chemotherapy. Although the biobank available included approximately 200 samples, only 58 were available for analysis. The presented workflow includes sample selection, experimental optimization, repeatability estimation, data preprocessing, data fusion, and feature selection. Specific difficulties have been the small number of samples and the skew distribution of the CR duration among the patients. Further, we had to deal with both noisy SELDI-MS data and a diverse patient cohort. This has been handled by sample selection and several methods for data preprocessing and feature detection in the analysis workflow. Four conceptually different methods for peak detection and alignment were considered, as well as two diverse methods for feature selection. The peak detection and alignment methods included the recently developed annotated regions of significance (ARS) method, the SELDI-MS software Ciphergen Express which was regarded as the standard method, segment-wise spectral alignment by a genetic algorithm (PAGA) followed by binning, and, finally, binning of raw data. In the feature selection, the "standard" Mann-Whitney t test was compared with a hierarchical orthogonal partial least-squares (O-PLS) analysis approach. The combined information from all these analyses gave a collection of 21 protein peaks. These were regarded as the most potential and robust biomarker candidates since they were picked out as significant features in several of the models. The chosen peaks will now be our first choice for the continuing work on protein identification and biological validation. The identification will be performed by chromatographic purification and MALDI MS/MS. Thus, we have shown that the use of several data handling methods can improve a protein profiling workflow from experimental optimization to a predictive model. The framework of this methodology should be seen as general and could be used with other one-dimensional spectral omics data than SELDI MS including an adequate number of samples.