Determination of methionine-enkephalin and leucine-enkephalin by LC-MS in human plasma: Study of pre-analytical stability.

The aim of this work was to assess the influence of preanalytical variables on the stability of two endogenous opioid peptides (Methionine-Enkephalin and Leucine-Enkephalin) in human plasma. For this purpose, first a sensitive LC-MS/MS analytical method was developed and validated for the simultaneous quantitative analysis of these two peptides. The methodology consisted of a simple protein precipitation step followed by UPLC separation and MRM quantitative analysis using a stable isotope labelled Methionine-Enkephalin as internal standard. The method with a limit of quantitation of 10 pg/mL showed good reproducibility with excellent accuracy and precision, and was linear up to 2000 pg/mL. An extensive evaluation of the pre-analytical stability of these peptides in human blood was carried out to ensure an adequate sample collection procedure to obtain reliable results in the analysis of clinical samples.

Simultaneous quantitative analysis of N-acylethanolamides in clinical samples.

A simple and rapid analytical method is described for the simultaneous quantitative analysis of three different N-acylethanolamides in human biological samples: anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA). The method is based on a new hybrid solid phase extraction-precipitation technology followed by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) analysis using d(4)-AEA as the internal standard. The method is linear up to 100ng/ml with a limit of quantitation of 50pg/ml for AEA and 100pg/ml for OEA and PEA. Good reproducibility, accuracy, and precision were demonstrated during the method validation. Application of this new methodology to the analysis of clinical study samples is presented.

MALDI-TOF serum protein profiling for the detection of breast cancer.

BACKGROUND: Proteomic expression profiling has been suggested as a potential tool for the early diagnosis of cancer and other diseases. The objective of our study was to assess the feasibility of this approach for the detection of breast cancer. MATERIALS AND METHODS: In a randomized block design pre-operative serum samples obtained from 78 breast cancer patients and 29 controls were used to generate high-resolution MALDI-TOF protein profiles. The spectra generated using C8 magnetic beads assisted mass spectrometry were smoothed, binned and normalized after baseline correction. Linear discriminant analysis with double cross-validation, based on principal component analysis, was used to classify the protein profiles. RESULTS: A total recognition rate of 99%, a sensitivity of 100%, and a specificity of 97.0% for the detection of breast cancer were shown. The area under the curve of the classifier was 98.3%, which demonstrates the separation power of the classifier. The first 2 principal components account for most of the between- group separation. CONCLUSIONS: Double cross-validation showed that classification could be attributed to actual information in the protein profiles rather than to chance. Although preliminary, the high sensitivity and specificity indicate the potential usefulness of serum protein profiles for the detection of breast cancer.

Detection of colorectal cancer using MALDI-TOF serum protein profiling.

Serum protein profiling is a promising approach for classification of cancer versus non-cancer samples. The objective of our study was to assess the feasibility of mass spectrometry based protein profiling for the discrimination of colorectal cancer (CRC) patients from healthy individuals. In a randomized block design, pre-operative serum samples obtained from 66 colorectal cancer patients and 50 controls were used to generate MALDI-TOF protein profiles. After pre-processing of the spectra, linear discriminant analysis with double cross-validation was used to classify protein profiles. A total recognition rate (92.6%), sensitivity (95.2%) and specificity (90.0%) for the detection of CRC were shown. The area under the curve of the classifier was 97.3%, and demonstrated the high, significant separation power of the classifier. Double cross-validation shows that classification can be attributed to information in the protein profile. Although preliminary, the high sensitivity and specificity indicate the potential usefulness of serum protein profiles for the detection of colorectal cancer.

Reliability of human serum protein profiles generated with C8 magnetic beads assisted MALDI-TOF mass spectrometry.

Protein profiling with mass spectrometry is a promising approach for classification and identification of biomarkers; however, there is debate about measurement quality and reliability. Here, we present a pipeline for preprocessing, statistical data analysis and presentation. Serum samples of 16 healthy individuals are used to generate protein profiles with high-resolution MALDI-TOF after isolation of peptides with C8 magnetic beads. Analysis of variance was performed after binning, baseline correction and normalization of the mean spectra. Relative variations in the spectra are expressed as coefficient of variation, which depending on the respective preanalytical variation parameter investigated, was found to range between 0.15 and 0.67 in this study. With this novel method, the reproducibility of our protein profiling procedure could be quantified. We showed that circadian rhythm and the number of freeze-thaw cycles had relatively limited influence on serum protein profiles, whereas the period between collection and serum centrifugation had a more pronounced effect.