A combined tryptic peptide and winged peptide internal standard approach for the determination of α‐lactalbumin in dairy products by ultra high performance liquid chromatography with tandem mass spectrometry

Lai, Shiyun; Zhang, Jingshun; Chen, Qi; Huang, Baifen; Ren, Yiping

doi:10.1002/jssc.201401279

Cited by 10 publications

(4 citation statements)

References 33 publications

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“…Scott et al investigated how the number of amino acids used for the extension in WiSIL peptides influences their digestion . The number of flanking amino acids in WiSIL peptides varies between studies, ,,− and an improved performance of peptides with longer extensions (6 amino acids) has been observed for WiSIL peptides and QConCATs, , especially if the sequence contains amino acids that are known to affect trypsin cleavage, such as Asp and Glu C- and N-terminal to Lys/Arg . The WiSIL peptides in our study were extended by two amino acids at the N- and C-terminus (Figure A), which could be a reason for the lack of improvement compared with the SIL approach.…”

Section: Results and Discussionmentioning

confidence: 79%

Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid

2017

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Absolute protein quantification by selected reaction monitoring (SRM, also MRM) is an alternative to immunoassays, and the gold standard here is the addition of stable-isotope labeled (SIL) proteins (PSAQ). Cerebrospinal fluid (CSF) is the preferred source of biomarkers for neurological diseases, and recent improvements in mass spectrometry enable the quantification of disease-relevant proteins in CSF. We used alpha-synuclein SRM to investigate alternatives to the PSAQ approach in human CSF regarding precision and accuracy, including SIL peptides, winged SIL (WiSIL) peptides, and quantitative protein epitope signature tags (QPrESTs). All approaches yielded precise results in CSF with CV values <15% in several runs for all four measured peptides. PSAQ and QPrEST also showed good accuracy (deviation ≤15%), whereas SIL and WiSIL peptides yielded deviations up to 54% that greatly depended on the measured peptide. Total protein concentration in CSF did not affect precision and accuracy. Thus, our study indicates that all four approaches are suitable for relative quantification of alpha-synuclein in CSF. QPrESTs are a valuable alternative to PSAQ in terms of precision and accuracy, although SIL and WiSIL peptides can yield accurate results as well when peptides are selected consciously.

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Section: Results and Discussionmentioning

confidence: 79%

Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid

2017

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“…These assumptions do not always hold true and the lost connection between protein and its peptides through digestion makes it more difficult to trace back to account for the errors. Even though bottom-up protein quantitation is quite mature, the creation of new standardization procedures to account for these limitations is still an active area of research [42]. Of course, the development of a specific application with the top-down approach on a triple quadrupole mass spectrometer would require comprehensive evaluation of sample preparation, sample enrichment (if necessary), and matrix effects that would be expected to alter quantitative performance from the idealized case presented in this paper.…”

Section: Resultsmentioning

confidence: 99%

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

Wang

Combe²,

Schug

2016

J. Am. Soc. Mass Spectrom.

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Abstract. Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostatespecific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

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“…4 ), when they were spiked into the mobile phase, indicating that using the internal standard peptides containing natural flanking sequences around the cleavage sites improves quantitative accuracy. Identical approaches using winged peptides as the internal standards were recently applied to measure human cytokine proteins by Scott et al [ 34 ], and bovine lactoferrin and α-lactalbumin in dairy products by Zhang et al [ 35 ] and Lai et al [ 36 ]. The good consistency suggests that winged peptides as the internal standards can best mimic the analytical behavior of intact OsGSTF14 and OsGSTU6 proteins.…”

Section: Resultsmentioning

confidence: 99%

Quantitation of glutathione S-transferases in rice (Oryza sativa L.) roots exposed to cadmium by liquid chromatography–tandem mass spectrometry using isotope-labeled wing peptides as an internal standard

et al. 2017

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BackgroundPlant glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional enzymes involved in heavy metal cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione to heavy metals. Previous studies demonstrated that individual rice GSTs were differentially induced by heavy metal exposure at the mRNA transcript level. However, little information is available concerning changes in protein concentration of rice GSTs under heavy metal stress. Because the correlation between changes in protein concentration and gene expression under abiotic stress is poor, direct determination of rice GSTs protein concentrations during cadmium (Cd) exposure is a more effective and reliable approach to explore possible mechanisms of rice Cd translocation and accumulation.ResultsThis study established an optimized and advanced liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based targeted proteomics assay for quantification of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots. The tryptic signature peptides were chosen as surrogate analytes and winged peptides containing the isotope-labeled signature peptides were used as the internal standards. The signature peptides exhibited good linearity in the range of 0.6–60 and 0.3–30 nM, respectively. The limit of detection and limit of quantification were 4.5 and 14.5 µg/g for OsGSTF14, respectively, and 2.1 and 7.0 µg/g for OsGSTU6. The spiking recoveries rates at low, medium and high levels were in the range of 72.5–93.4%, with intra- and inter-day precisions of 5.5–9.1 and 4.2–10.2%, respectively.ConclusionsThe assay successfully quantified the temporal and dose responses of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots, with good accuracy, precision and high-throughput. This assay will have significant application in developing quantification methods of other proteins in Cd-stressed rice, which may provide more insight into the mechanisms of Cd translocation and accumulation in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0214-2) contains supplementary material, which is available to authorized users.

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A combined tryptic peptide and winged peptide internal standard approach for the determination of α‐lactalbumin in dairy products by ultra high performance liquid chromatography with tandem mass spectrometry

Cited by 10 publications

References 33 publications

Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid

Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

Quantitation of glutathione S-transferases in rice (Oryza sativa L.) roots exposed to cadmium by liquid chromatography–tandem mass spectrometry using isotope-labeled wing peptides as an internal standard

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