Quantification of intact human insulin‐like growth factor‐I in serum by nano‐ultrahigh‐performance liquid chromatography/tandem mass spectrometry

Lopes, Filipe; Cowan, David; Thevis, Mario; Thomas, Andreas; Parkin, Mark C.

doi:10.1002/rcm.6908

Cited by 24 publications

(30 citation statements)

References 27 publications

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“…A simple pellet digestion protocol (Becher et al, 2017) was used here for removal of potentially interfering matrix components such as small molecules, phospholipids, peptides (Ouyang et al, 2012) and the added Triton X-100 surfactant which could otherwise have a dramatic impact on MS sensitivity (Cox and Emili, 2006). Considering the molecular weight difference between C9-S and C9-L, i.e., 25 and 54 kDa, and the potential solubility of smaller proteins in organic precipitation solvents (Lopes et al, 2014), we ascertained the equivalent recovery for both isoforms, ensuring accurate quantification. The signal observed for both C9-S and C9-L unique peptides in transfected HEK293 cells further confirmed the correct extraction and precipitation of C9ORF72 isoforms.…”

Section: Discussionmentioning

confidence: 99%

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

et al. 2018

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Frontotemporal dementia (FTD) is a fatal neurodegenerative disease characterized by behavioral and language disorders. The main genetic cause of FTD is an intronic hexanucleotide repeat expansion (G4C2)n in the C9ORF72 gene. A loss of function of the C9ORF72 protein associated with the allele-specific reduction of C9ORF72 expression is postulated to contribute to the disease pathogenesis. To better understand the contribution of the loss of function to the disease mechanism, we need to determine precisely the level of reduction in C9ORF72 long and short isoforms in brain tissue from patients with C9ORF72 mutations. In this study, we developed a sensitive and robust mass spectrometry (MS) method for quantifying C9ORF72 isoform levels in human brain tissue without requiring antibody or affinity reagent. An optimized workflow based on surfactant-aided protein extraction and pellet digestion was established for optimal recovery of the two isoforms in brain samples. Signature peptides, common or specific to the isoforms, were targeted in brain extracts by multiplex MS through the parallel reaction monitoring mode on a Quadrupole–Orbitrap high resolution mass spectrometer. The assay was successfully validated and subsequently applied to frontal cortex brain samples from a cohort of FTD patients with C9ORF72 mutations and neurologically normal controls without mutations. We showed that the C9ORF72 short isoform in the frontal cortices is below detection threshold in all tested individuals and the C9ORF72 long isoform is significantly decreased in C9ORF72 mutation carriers.

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Section: Discussionmentioning

confidence: 99%

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

et al. 2018

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“…[1] Most existing analytical procedures to determine these target peptides in blood or urine employ liquid chromatography coupled to mass spectrometry (LC-MS) due to its distinguished specificity. [2,[5][6][7][8][9][10][11][12][13][14][15][16][17][18] The principle of combining several of these compounds into one screening method was also realized recently and has already been implemented into routine doping controls of important sport events. [8,17,19] Methods based on bioassays (ELISA, RIA, etc.)…”

Section: Introductionmentioning

confidence: 99%

Expanded test method for peptides >2 kDa employing immunoaffinity purification and LC‐HRMS/MS

Thomas

Walpurgis

Tretzel

et al. 2015

Drug Testing and Analysis

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Bioactive peptides with an approximate molecular mass of 2-12 kDa are of considerable relevance in sports drug testing. Such peptides have been used to manipulate several potential performance-enhancing processes in the athlete's body and include for example growth hormone releasing hormones (sermorelin, CJC-1293, CJC-1295, tesamorelin), synthetic/animal insulins (lispro, aspart, glulisine, glargine, detemir, degludec, bovine and porcine insulin), synthetic ACTH (synacthen), synthetic IGF-I (longR(3) -IGF-I) and mechano growth factors (human MGF, modified human MGF, 'full-length' MGF). A combined initial test method using one analytical procedure is a desirable tool in doping controls and related disciplines as requests for higher sample throughput with utmost comprehensiveness preferably at reduced costs are constantly issued. An approach modified from an earlier assay proved fit-for-purpose employing pre-concentration of all target analytes by means of ultrafiltration, immunoaffinity purification with coated paramagnetic beads, nano-ultra high performance liquid chromatography (UHPLC) separation, and subsequent detection by means of high resolution tandem mass spectrometry. The method was shown to be applicable to blood and urine samples, which represent the most common doping control specimens. The method was validated considering the parameters specificity, recovery (11-69%), linearity, imprecision (<25%), limit of detection (5-100 pg in urine, 0.1-2 ng in plasma), and ion suppression. The analysis of administration study samples for insulin degludec, detemir, aspart, and synacthen provided the essential data for the proof-of-principle of the method.

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“…Utilization of non-tapered emitters with an internal diameter in the range of 10-30 mm provides an adequate compromise between sensitivity, system robustness, and sample consumption. [97][98][99][100][101] Wider internal diameters reduce potential clogging and facilitate more stable ionization spray, resulting in more reproducible mass spectra and ion chromatograms.…”

Section: Mass Spectrometry Data Acquisitionmentioning

confidence: 99%

Single-platform ‘multi-omic’ profiling: unified mass spectrometry and computational workflows for integrative proteomics–metabolomics analysis

2018

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The objective of omics studies is to globally measure the different classes of cellular biomolecules present in a biological specimen (e.g. proteins, metabolites) as accurately as possible in order to investigate the corresponding 'states' of biological systems. High throughput omics technologies are emerging as an increasingly powerful toolkit in the rapidly advancing field of systems biology, enabling the systematic study of dynamic molecular processes that drive core cell functions like growth, sensing, and environmental adaptation. Advances in high resolution mass spectrometry, in particular, now allow for the near comprehensive study of cellular proteins and metabolites that underlie physiological homeostasis and disease pathogenesis. Yet while the expression levels, modification states, and functional associations of diverse molecular species are now measurable, existing proteomic and metabolomic data generation and analysis workflows are often specialized and incompatible. Hence, while there are now many reports of ad hoc combinations of unimolecular proteomic and metabolomic workflows, only a limited number of multi-omic profiling approaches have been reported for obtaining different molecular measurements (proteins, metabolites, nucleic acids) in parallel from a single biological sample. Moreover, elucidating how the myriad of measured cellular components are linked together functionally within the metabolic processes, signal transduction pathways, and macromolecular interaction networks central to living systems remains a massive, complicated, and uncertain endeavor. Presented here is a review of convergent mass spectrometry-based multi-omic methodologies, with a focus on notable recent advances and remaining challenges in terms of efficient sample preparation, biochemical separations, data acquisition, and integrative computational strategies. We outline a unifying network-based integrative framework to better derive biological knowledge from integrated profiling studies with the goal of realizing the full potential of multi-omic data sets.

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Quantification of intact human insulin‐like growth factor‐I in serum by nano‐ultrahigh‐performance liquid chromatography/tandem mass spectrometry

Cited by 24 publications

References 27 publications

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

Expanded test method for peptides >2 kDa employing immunoaffinity purification and LC‐HRMS/MS

Single-platform ‘multi-omic’ profiling: unified mass spectrometry and computational workflows for integrative proteomics–metabolomics analysis

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