An Empirical Approach to Signature Peptide Choice for Selected Reaction Monitoring: Quantification of Uromodulin in Urine

Fu, Qin; Grote, Eric; Zhu, Jie; Jelinek, Christine; Köttgen, Anna; Coresh, Josef; Eyk, Jennifer E. Van

doi:10.1373/clinchem.2015.242495

Cited by 21 publications

(30 citation statements)

References 27 publications

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“…Differential interindividual variability of proteins was observed, indicating unique regulation (e.g., mediated by individual genetic and epigenetic factors) of these proteins. The highest variability was observed for Tamm-Horsfall protein, consistent with reported values (Fu et al, 2016). However, as the main aim of this report was methodological, unique interindividual variability has no effect on our conclusions.…”

Section: Discussionsupporting

confidence: 90%

An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics

Vrana

Goodling

Afkarian

et al. 2016

Drug Metabolism and Disposition

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The existing biobanks of remnant tissue from clinically indicated kidney biopsies are attractive potential reservoirs for quantification of clinically relevant human tissue proteins by quantitative proteomics. However, a significant caveat of this strategy is that the tissues are often preserved in optimal cutting temperature (OCT) medium. Although OCT is an effective method of preserving the morphologic and immunohistological characteristics of tissues for later study, it significantly impacts efforts to quantify protein expression by liquid chromatography-tandem mass spectrometry methods. We report here a simple, reproducible, and cost-effective procedure to extract proteins from OCT-embedded tissue samples. Briefly, the excess frozen OCT medium was scraped before thawing from the tissue specimens stored at -80°C for ∼3 months. The tissue samples were homogenized and diethyl ether/methanol extraction was performed to remove the remaining OCT medium. The recovered protein was denatured, reduced, and alkylated. The second step of protein extraction and desalting was performed by chloroform/methanol/ water extraction of denatured proteins. The resultant protein pellet was trypsin-digested and the marker proteins of various kidney cellular compartments were quantified by targeted selective reaction monitoring proteomics. Upon comparison of peptide signals from OCT-embedded tissue and flash-frozen tissue from the same donors, both individual protein quantities, and their interindividual variabilities, were similar. Therefore, the approach reported here can be applied to clinical reservoirs of OCT-preserved kidney tissue to be used for quantitative proteomics studies of clinically relevant proteins expressed in different parts of the kidney (including drug transporters and metabolizing enzymes).

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Section: Discussionsupporting

confidence: 90%

An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics

Vrana

Goodling

Afkarian

et al. 2016

Drug Metabolism and Disposition

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show abstract

“…Following identification of potential proteomic biomarkers using top-down/bottom-up mass spectrometry, validation and absolute quantification of urine biomarkers is best achieved by Selected Reaction Monitoring (SRM) [35, 78, 79]. SRM, also known as Multiple Reaction Monitoring (MRM), allows previously defined peptides and fragment ions, known as signature peptides, to be quantified by monitoring multiple fragment ions produced during collision-induced dissociation [18].…”

Section: Advancements In Mass Spectrometry Based Urinary Proteomicsmentioning

confidence: 99%

“…SRM, also known as Multiple Reaction Monitoring (MRM), allows previously defined peptides and fragment ions, known as signature peptides, to be quantified by monitoring multiple fragment ions produced during collision-induced dissociation [18]. For robust SRM analysis with strong signal intensity, the peptides for SRM analysis must be unique to the protein of interest, should not contain post-translational modifications, and should not have genetically encoded variations [78]. Absolute quantification can be performed with stable isotope-labeled peptides corresponding to the signature peptides.…”

Section: Advancements In Mass Spectrometry Based Urinary Proteomicsmentioning

confidence: 99%

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Current state of the art for enhancing urine biomarker discovery

Harpole

Davis

Espina

2016

Expert Review of Proteomics

113

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Urine is a highly desirable biospecimen for biomarker analysis because it can be collected recurrently by non-invasive techniques, in relatively large volumes. Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect, at a given time point, an individual's metabolic and pathophysiologic state. High-resolution mass spectrometry, coupled with state of the art fractionation systems are revealing the plethora of diagnostic/prognostic proteomic information existing within urinary exosomes, glycoproteins, and proteins. Affinity capture pre-processing techniques such as combinatorial peptide ligand libraries and biomarker harvesting hydrogel nanoparticles are enabling measurement/identification of previously undetectable urinary proteins. Future challenges in the urinary proteomics field include a) defining either single or multiple, universally applicable data normalization methods for comparing results within and between individual patients/data sets, and b) defining expected urinary protein levels in healthy individuals.

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“…These include the proper collection, processing and storage of specimens from large cohorts for proteomic analysis, development of standard operating procedures for multiple reaction monitoring, external quality control for assays including blind duplicates, and proficiency samples from quality control pools [17]. Substantial progress has been made in developing multiple reaction monitoring assays in urine, particularly of the candidate CKD biomarker uromodulin [22,23]. While CKD BioCon has not produced any breakthrough CKD biomarkers to date, it clearly has set standards for programmatic efforts to identify renal disease biomarkers and raised standards to those used in successful cancer biomarker consortiums.…”

Section: Difficult Lessons Learnedmentioning

confidence: 99%

Applying proteomics to detect early signs of chronic kidney disease: where has the magic gone?

Klein

2017

Expert Review of Proteomics

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An Empirical Approach to Signature Peptide Choice for Selected Reaction Monitoring: Quantification of Uromodulin in Urine

Cited by 21 publications

References 27 publications

An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics

An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics

Current state of the art for enhancing urine biomarker discovery

Applying proteomics to detect early signs of chronic kidney disease: where has the magic gone?

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