Comparison of normalisation methods for surface-enhanced laser desorption and ionisation (SELDI) time-of-flight (TOF) mass spectrometry data

<p>Abstract</p> <p>Background</p> <p>Mass spectrometry for biological data analysis is an active field of research, providing an efficient way of high-throughput proteome screening. A popular variant of mass spectrometry is SELDI, which is often used to measure sample populations with the goal of developing (clinical) classifiers. Unfortunately, not only is the data resulting from such measurements quite noisy, variance between replicate measurements of the same sample can be high as well. Normalisation of spectra can greatly reduce the effect of this technical variance and further improve the quality and interpretability of the data. However, it is unclear which normalisation method yields the most informative result.</p> <p>Results</p> <p>In this paper, we describe the first systematic comparison of a wide range of normalisation methods, using two objectives that should be met by a good method. These objectives are minimisation of inter-spectra variance and maximisation of signal with respect to class separation. The former is assessed using an estimation of the coefficient of variation, the latter using the classification performance of three types of classifiers on real-world datasets representing two-class diagnostic problems. To obtain a maximally robust evaluation of a normalisation method, both objectives are evaluated over multiple datasets and multiple configurations of baseline correction and peak detection methods. Results are assessed for statistical significance and visualised to reveal the performance of each normalisation method, in particular with respect to using no normalisation. The normalisation methods described have been implemented in the freely available MASDA R-package.</p> <p>Conclusion</p> <p>In the general case, normalisation of mass spectra is beneficial to the quality of data. The majority of methods we compared performed significantly better than the case in which no normalisation was used. We have shown that normalisation methods that scale spectra by a factor based on the dispersion (e.g., standard deviation) of the data clearly outperform those where a factor based on the central location (e.g., mean) is used. Additional improvements in performance are obtained when these factors are estimated locally, using a sliding window within spectra, instead of globally, over full spectra. The underperforming category of methods using a globally estimated factor based on the central location of the data includes the method used by the majority of SELDI users.</p

Validation of previously identified serum biomarkers for breast cancer with SELDI-TOF MS: a case control study

<p>Abstract</p> <p>Background</p> <p>Serum protein profiling seems promising for early detection of breast cancer. However, the approach is also criticized, partly because of difficulties in validating discriminatory proteins. This study's aim is to validate three proteins previously reported to be discriminative between breast cancer cases and healthy controls. These proteins had been identified as a fragment of inter-alpha trypsin inhibitor H4 (4.3 kDa), C-terminal-truncated form of C3a des arginine anaphylatoxin (8.1 kDa) and C3a des arginine anaphylatoxin (8.9 kDa).</p> <p>Methods</p> <p>Serum protein profiles of 48 breast cancer patients and 48 healthy controls were analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Differences in protein intensity between breast cancer cases and controls were measured with the Mann-Whitney U test and adjusted for confounding in a multivariate logistic regression model.</p> <p>Results</p> <p>Four peaks, with mass-to-charge ratio (<it>m/z</it>) 4276, 4292, 8129 and 8941, were found that were assumed to represent the previously reported proteins. <it>M/</it>z 4276 and 4292 were statistically significantly decreased in breast cancer cases compared to healthy controls (p < 0.001). M/<it>z </it>8941 was decreased in breast cancer cases (p < 0.001) and <it>m/z </it>8129 was not related with breast cancer (p = 0.87). Adjustment for sample preparation day, sample storage duration and age did not substantially alter results.</p> <p>Conclusion</p> <p><it>M/z </it>4276 and 4292 both represented the previously reported 4.3 kDa protein and were both decreased in breast cancer patients, which is in accordance with the results of most previous studies. <it>M/z </it>8129 was in contrast with previous studies not related with breast cancer. Remarkably, <it>m/z </it>8941 was decreased in breast cancer cases whereas in previous studies it was increased. Differences in patient populations and pre-analytical sample handling could have contributed to discrepancies. Further research is needed before we can conclude on the relevance of these proteins as breast cancer biomarkers.</p

Mass spectrometry protein expression profiles in colorectal cancer tissue associated with clinico-pathological features of disease

Background: Studies of several tumour types have shown that expression profiling of cellular protein extracted from surgical tissue specimens by direct mass spectrometry analysis can accurately discriminate tumour from normal tissue and in some cases can sub-classify disease. We have evaluated the potential value of this approach to classify various clinico-pathological features in colorectal cancer by employing matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS). Methods: Protein extracts from 31 tumour and 33 normal mucosa specimens were purified, subjected to MALDI-Tof MS and then analysed using the `GenePattern' suite of computational tools (Broad Institute, MIT, USA). Comparative Gene Marker Selection with either a t-test or a signal-to-noise ratio (SNR) test statistic was used to identify and rank differentially expressed marker peaks. The k-nearest neighbours algorithm was used to build classification models either using separate training and test datasets or else by using an iterative, `leave-one-out' cross-validation method. Results: 73 protein peaks in the mass range 1800-16000Da were differentially expressed in tumour verses adjacent normal mucosa tissue (P <= 0.01, false discovery rate <= 0.05). Unsupervised hierarchical cluster analysis classified most tumour and normal mucosa into distinct cluster groups. Supervised prediction correctly classified the tumour/normal mucosa status of specimens in an independent test spectra dataset with 100\% sensitivity and specificity (95\% confidence interval: 67.9-99.2\%). Supervised prediction using `leave-one-out' cross validation algorithms for tumour spectra correctly classified 10/13 poorly differentiated and 16/18 well/moderately differentiated tumours (P = < 0.001; receiver-operator characteristics - ROC - error, 0.171); disease recurrence was correctly predicted in 5/6 cases and disease-free survival (median follow-up time, 25 months) was correctly predicted in 22/23 cases (P = < 0.001; ROC error, 0.105). A similar analysis of normal mucosa spectra correctly predicted 11/14 patients with, and 15/19 patients without lymph node involvement (P = 0.001; ROC error, 0.212). Conclusions: Protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value in studies aimed at improved molecular classification of this disease. Further studies, with longer follow-up times and larger patient cohorts, that would permit independent validation of supervised classification models, would be required to confirm the predictive value of tumour spectra for disease recurrence/patient survival

Chaperonin Hsp60 and Cancer Therapies

The heat shock protein 60 (Hsp60) is a chaperonin belonging to the chaperoning (chaperone) system that typically contributes to protein homeostasis inside mitochondria, but also plays various non-canonical roles unrelated to protein quality control beyond the organelle. Chaperonopathies are disorders in which chaperones play an etiologic-pathogenic role and contribute to the onset/progression of disease. Hsp60 chaperonopathies by mistake are diseases in which the chaperonin is apparently normal (as far as it can be determined with current methodologies) but it actively contributes to pathology, for example in certain types of cancer, and autoimmune and chronic inflammatory disorders. In certain cancers, Hsp60 is associated with the onset of malignancy and metastasization, although the mechanisms are poorly understood. In this chapter, we summarize findings on Hsp60 quantitative changes and distribution alterations in cells and tissues accompanying tumor initiation and progression. We also discuss the potential of HSP60-based anticancer therapies that are currently being investigated.Methods Journals and data bases were surveyed, and pertinent works were chosen for discussion. Results The data have stimulated experimental and clinical studies aiming at establishing the usefulness of Hsp60 as biomarker for diagnosis, and for assessing prognosis and response to treatment. Likewise, investigations are ongoing on the possible use of Hsp60 as a therapeutic agent or target. The reported results indicate that in neoplasms, Hsp60 migrates outside its canonical location, the mitochondrion, augments in the cytoplasm and plasma-cell membrane, and exits the cell via lipid rafts-exosomes. Exosomal Hsp60 occurs in extracellular fluids such as blood, through which it reaches target cells near and far, normal or cancerous. With these target cells, exosomes carrying Hsp60 and other molecules interact and, thereby, modify their functions. Thus, detection of exosomal Hsp60 in body fluids appears as a promising variety of liquid biopsy applicable to monitoring cancers already diagnosed and to screen for malignancies before they are clinically manifest. We also discuss Hsp60-based vaccines as a novel means of eliminating cancer cells with cytotoxic T lymphocytes (CTL). Tumor-derived Hsp60 associated with a tumorderived antigen activates CD8+ T cells and induces an antitumor immune response. It is highly probable that soon there will be implementation of clinical trials, involving the use of Hsp60 alone or in various combinations and complexes to prevent cancer progression and treat patients. Conclusions Hsp60 is actively involved in tumor development and progression. Its presence in extracellular fluids renders it a potential non-invasive biomarker. Also, considering the antitumor activities of Hsp60 observed in some types of cancer one can foresee a bright future for Hsp60-based therapies