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In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of LC - triple quadrupole MS-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase LC - triple quadrupole MS method for analysis of cancer-related intact proteins was evaluated. Seven growth factors (5.5 - 26.5 kDa; isoelectric points: 4.6 - 9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% (v/v) difluoroacetic acid as a mobile phase modifier. The increase of MS sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole MS instrumentation, which could be beneficial in many application fields. This article is protected by copyright. All rights reserved.
This article was published in the following journal.
Name: Journal of separation science
Gangliosides (GG) are glycosphingolipids, composed of a ceramide moiety (fatty acid and long chain base) linked to an oligosaccharide chain containing one (or more) molecule of sialic acid. After lipi...
A rapid, sensitive and widely applicable method for quantitative analysis of underivatized amino acids in different biological matrices by ultra-performance liquid chromatography-tandem mass spectrometry.
A rapid, sensitive and widely applicable method for the simultaneous quantitative analysis of 20 underivatized amino acids in different biological matrices, including serum, plasma and tissue homogena...
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Liquid chromatography-mass spectrometry (LC-MS) is one of the most important analytical chemistry techniques for the detection and characterization of biologically active compounds of low abundance-fo...
The purpose of the study is to develop a method for detecting, isolating and quantifying lamivudine in biological substances. Lamivudine was isolated by liquid-liquid and solid phase extraction. The c...
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Fractionation of a vaporized sample as a consequence of partition between a mobile gaseous phase and a stationary phase held in a column. Two types are gas-solid chromatography, where the fixed phase is a solid, and gas-liquid, in which the stationary phase is a nonvolatile liquid supported on an inert solid matrix.
A method of separation of two or more substances by repeated distribution between two immiscible liquid phases that move past each other in opposite directions. It is a form of liquid-liquid chromatography. (Stedman, 25th ed)
Chromatographic techniques in which the mobile phase is a liquid.
Tabular numerical representations of sequence motifs displaying their variability as likelihood values for each possible residue at each position in a sequence. Position-specific scoring matrices (PSSMs) are calculated from position frequency matrices.
Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed.
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Bladder Cancer Brain Cancer Breast Cancer Cancer Cervical Cancer Colorectal Head & Neck Cancers Hodgkin Lymphoma Leukemia Lung Cancer Melanoma Myeloma Ovarian Cancer Pancreatic Cancer ...