A new quantification method for assessing plasma concentrations of pemetrexed and its polyglutamate metabolites.

Currently no quantification method exists for potentially therapeutically relevant polyglutamate metabolites of the drug pemetrexed which is used for the treatment of lung carcinoma patients. We developed and tested an LC-MS/MS-based analytical assay that uses isotope-labeled internal standards to quantify pemetrexed and its (poly)glutamate metabolites in clinical human plasma samples of lung carcinoma patients. UHPLC chromatography and triple quadrupole mass spectrometry showed an LLOQ of 0.2nmol/L for pemetrexed and an LLOQ of 0.5nmol/L for the two metabolites (one glutamate and two glutamate moieties covalently bound to the pemetrexed molecule, for which no other quantification methods have previously been published). The recoveries for PMTX and its metabolites ranged between 30% and 67%. Precision and accuracy at a concentration of 20nmol/L for all four analytes was well below 15% CV. The precision (RSD) in the biological replicates of the separate days (within-run precision) as well as the reproducibility over several days (between-run precision), tested in the range of 5-250nmol/L, were all below 15%. Autosampler, benchtop and freeze-thaw cycle stability of the analytes was also demonstrated. To illustrate the new assay in a relevant biological context, concentrations of pemetrexed and the two metabolites were quantified in plasma samples of lung carcinoma patients treated with pemetrexed. The assay is straightforward, relatively easy to perform, and has potential for use in therapeutic drug monitoring in non-small cell lung carcinoma patients.

Proteomics urine analysis of pregnant women suffering from multiple sclerosis.

Multiple sclerosis (MScl) frequently is remitted during the third trimester of pregnancy but exacerbated in the first postpartum period. In this context, we investigated protein identification, its abundance, and its change in urine related to these two periods. Using mass spectrometry (LTQ Orbitrap), we identified 1699 tryptic peptides (related to 402 proteins) in urine from 31 MScl and 8 control at these two periods. Pregnancy-related peptides were significantly elevated (p < 0.01) in MScl patients compared with controls (Analysis 1: 531 peptides in MScl and 36 peptides in controls higher abundant in the third trimester compared to postpartum). When comparing the longitudinal differences (Analysis 2), we identified 43 (related to 35 proteins) MScl disease-associated peptides (p < 0.01) with increased or decreased difference ratio in MScl compared with controls. The most discriminating peptides identified were trefoil factor 3 and lysosomal-associated membrane protein 2. Both proteins have a role in the innate immune system. Three proteins with a significant decreased ratio were plasma glutamate carboxypeptidase, Ig mu chain C region, and osteoclast associated immune like receptor. Our results indicate that the protein expression pattern in urine of MScl patients contains information about remote CNS and brain disease processes.

Comparative study of targeted and label-free mass spectrometry methods for protein quantification.

We compared data acquired on an LTQ-Orbitrap MS used in a typical shotgun proteomics setting (optimized for protein identification) with data from a quadrupole ion trap MS operated in the MRM mode. Six relative abundant proteins were quantified in identical sets of serum and CSF samples by the following methods: a qual/quant method with and without use of internal standards and a quantitative method (MRM with use of internal standards). Comparison of these methods with an antibody-based method in CSF samples showed good linearity for both methods (R(2) of 0.961 and 0.971 for the qual/quant method with use of internal standards and the quantitative method, respectively). Besides its better linearity, the quantitative method was also more reproducible with lower CVs for all samples. Next to these comparisons we also explored why a qual/quant approach had typically a lower reproducibility compared to MRM analyses. We observed that modified peptides, or peptides with a cysteine or a methionine, yielded a significant increase in CV. Furthermore, a positive correlation was found between the length of the peptide and the CV. We conclude that qual/quant is an alternative for the quantification of abundant proteins and that the use of internal standards in qual/quant could be advantageous. Furthermore, the ongoing development in MS techniques increases the possibilities of qual/quant in protein quantification.

The impact of delayed storage on the measured proteome and metabolome of human cerebrospinal fluid.

BACKGROUND: Because cerebrospinal fluid (CSF) is in close contact with diseased areas in neurological disorders, it is an important source of material in the search for molecular biomarkers. However, sample handling for CSF collected from patients in a clinical setting might not always be adequate for use in proteomics and metabolomics studies. METHODS: We left CSF for 0, 30, and 120 min at room temperature immediately after sample collection and centrifugation/removal of cells. At 2 laboratories CSF proteomes were subjected to tryptic digestion and analyzed by use of nano-liquid chromatography (LC) Orbitrap mass spectrometry (MS) and chipLC quadrupole TOF-MS. Metabolome analysis was performed at 3 laboratories by NMR, GC-MS, and LC-MS. Targeted analyses of cystatin C and albumin were performed by LC-tandem MS in the selected reaction monitoring mode. RESULTS: We did not find significant changes in the measured proteome and metabolome of CSF stored at room temperature after centrifugation, except for 2 peptides and 1 metabolite, 2,3,4-trihydroxybutanoic (threonic) acid, of 5780 identified peptides and 93 identified metabolites. A sensitive protein stability marker, cystatin C, was not affected. CONCLUSIONS: The measured proteome and metabolome of centrifuged human CSF is stable at room temperature for up to 2 hours. We cannot exclude, however, that changes undetectable with our current methodology, such as denaturation or proteolysis, might occur because of sample handling conditions. The stability we observed gives laboratory personnel at the collection site sufficient time to aliquot samples before freezing and storage at -80 °C.

The effect of preanalytical factors on stability of the proteome and selected metabolites in cerebrospinal fluid (CSF).

To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples. The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.

Quantitative matrix-assisted laser desorption ionization-fourier transform ion cyclotron resonance (MALDI-FT-ICR) peptide profiling and identification of multiple-sclerosis-related proteins.

We introduce a matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FT-ICR) method for quantitative peptide profiling, using peak height as a measure for abundance. Relative standard deviations in peak height of peptides spiked over 3 orders of magnitude in concentration were below 10% and allowed for accurate comparisons between multiple sclerosis and controls. Application on a set of 163 cerebrospinal fluid (CSF) samples showed significantly differential abundant peptides, which were subsequently identified into proteins (e.g., chromogranin A, clusterin, and complement C3).

The rate of false positive sequence matches of peptides profiled by MALDI MS and identified by MS/MS.

In most MALDI peptide profiling cases, sequencing is required to identify peptides of interest, preferentially by using different mass spectrometry techniques. Using identical samples, we determined the number of false positive matches in sequence of peptide identification using different mass spectrometers. This paper demonstrates that the reliability of the identification phase greatly benefits from concerted MS-technologies and determines the influence of mass accuracy, signal-to-noise and statistical score on peptide identification.