Optimization of liquid chromatography–multiple reaction monitoring cubed mass spectrometry assay for protein quantification: Application to aquaporin-2 water channel in human urine

Multiplexed assay for protein quantitation in the invertebrate Gammarus fossarum by liquid chromatography coupled to tandem mass spectrometry

“…The decrease in cellulase levels in the pre-molt stage D2 is also in accordance with previous observations in G. fossarum [13], which reported a peak of digestive enzymatic activity concomitant to energy acquisition in the early stages of the molt cycle, followed by a gradual decrease during the inter-and pre-molt stages, in relation to copular behavior.…”

“…However, this approach was not retained as the sensitivity of the assay was not high enough for all peptides. Another method used in our lab to reduce or eliminate matrix effects include optimizing sample preparation to remove interfering compounds from the samples [13], changing chromatographic parameters to avoid coelution of analytes, and interfering compounds to reduce the occurrence of matrix effects in the ion source [12]. Such methods were not retained.…”

“…Heavy labeled isotopologs of the targeted peptides are concomitantly monitored to ensure precise and accurate measurements [10,11]. Although not as sensitive as the most sensitive ELISAs, SRM routinely detects concentrations in the low to sub-nanogram per milliliter range after target peptide or protein enrichment or after extensive sample fractionation to reduce the likelihood of co-eluted interferences [12][13][14][15][16].…”

The clinical utility of mass spectrometry based protein assays

“…In multiple reports, LC-MS methods have been proposed and demonstrated for the measurement of safety biomarkers associated with acute toxicity or injury. Podocin and Podocalyxin have been proposed as measures of kidney glomerular injury [74][75][76]. LC-MS assays for pro-glucagon derived peptides such as GLP-1 [77], glucagon [11,78,79] and oxyntomodulin have been developed to overcome specificity and sensitivity challenges of traditional immunoassays [80].…”

Quantitation of Human Peptides and Proteins Via MS: Review of Analytically Validated Assays

“…Two alternative MRMbased techniques were developed to address this challenge: multiple reaction monitoring cubed (MRM3) and high-pressure, highresolution separations with intelligent selection and multiplexing (PRISM). MRM3 [3,4] includes an additional fragmentation step, which reduces inferences, improves signal-tonoise ratio and leads to an improved lowerlimit-of-quantitation (LLOQ). PRISM [5,6] fractionates a complex peptide mixture using high resolution reverse phase LC with online multiplexing MRM monitoring, adding Although MRM is an intrinsically sensitive MS technique, it is a unit mass resolution based approach and is prone to interference from background ions.…”