In this report, we discuss key issues for the successful application of MALDI-TOF mass spectrometry to quantify drugs. These include choice and preparation of matrix, nature of cationization agent, ...automation, and data analysis procedures. The high molecular weight matrix meso-tetrakis(pentafluorophenyl)porphyrin eliminates chemical noise in the low-mass range, a “brushing” spotting technique in combination with prestructured target plates enables fast preparation of homogeneous matrix crystals, and addition of Li+ leads to intense cationized drug species. Complex biological samples were cleaned up using a 96-well solid-phase extraction plate, and the purified samples were automatically spotted by a pipetting robot. To obtain a suitable data analysis procedure for the quantitative analysis of drugs by MALDI-TOF mass spectrometry, various data processing parameters were evaluated on our two model drugs lopinavir and ritonavir. Finally, and most importantly, it is shown that the above-described procedure can be successfully applied to quantify clinically relevant concentrations of lopinavir, an HIV protease inhibitor, in extracts of small numbers of peripheral blood mononuclear cells (1 × 106).
Relative affinity measurements of monovalent metal ions (M=Li+, Na+, Cu+ and Ag+) toward aliphatic nitriles have been performed using the kinetic method by dissociation of metal-bound dimer ions of ...the type R1C≡N-M+-N≡CR2. It was found, particularly for Cu+ and Ag+, that the affinity toward nitriles having long chains (>C6) is markedly enhanced. This is attributed to a bidentate interaction of the metal ion with the nitrile moiety and the aliphatic chain. Theoretical calculations on the copper complexes show that these bidentate structures enjoy about 30% greater copper-ion affinities compared to their linear counterparts. Such aliphatic interactions also play a major role in the dissociation chemistry of copper-bound tetramers of the kind (RC≡N)4Cu2+• where the long aliphatic chain R curls around the copper ion to facilitate electron transfer or a redox reaction to produce (RC≡N)2Cu+ + RC≡N+• + RC≡N.
Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography-mass spectrometry in concert with calcium-directed collision-induced ...dissociations. Our method does not require any modifications to the liquid chromatography-mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide.
Immunodepletion of high-abundance proteins from serum is a widely used initial step in biomarker discovery studies. In the present work we have investigated the reproducibility of the depletion step ...by comparing 250 serum samples from prostate cancer patients. All samples were depleted on a single immunoaffinity column over a time period of 6 weeks with automated peak detection and fraction collection. Reproducibility in terms of surface area of the depleted serum protein peak at 280
nm was below 7% relative standard deviation (R.S.D.) and the collected volume of the relevant fraction was 0.97
mL (4.5% R.S.D.). Proteins in the depleted serum fraction were subsequently digested with trypsin and analyzed by MALDI-FT-MS. The degree of the depletion of albumin, transferrin and alpha-1-antitrypsin was determined by comparing the intensity of peptide peaks before and after depletion of 11 samples taken at regular time intervals from amongst the 250 depleted, randomized samples. As a positive control we evaluated peaks of apolipoprotein A1 (the most abundant serum protein remaining after depleteion) showing a clear increase in intensity of these peaks in the depleted samples. From this study we conclude that the depletion of the 250 serum samples was complete and reproducible over a period of 6 weeks.
Tuberous sclerosis complex (TSC) is an autosomal dominant benign tumour syndrome caused by mutations to either the
TSC1 or
TSC2 tumour suppressor gene. The
TSC1 and
TSC2 gene products, TSC1 and TSC2, ...form a protein complex that integrates inputs from multiple signalling cascades to inactivate the small GTPase rheb, and thereby inhibit mTOR-dependent cell growth. We have used matrix-assisted laser desorption/ionisation time-of-flight and Fourier transform mass spectrometry to identify TSC1 and TSC2 phosphorylation sites and candidate TSC1 and TSC2 interacting proteins. We identified three sites of TSC2 phosphorylation and a novel site of TSC1 phosphorylation, and investigated the roles of these sites in regulating the activity of the TSC1–TSC2 complex. In addition, we identified three TSC1–TSC2 interacting proteins, including DOCK7 a putative rhebGEF.
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