Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum

Abstract

Abstract. We describe a systematic comparison of high and low resolution LC-MS/ MS assays for quantification of 25-hydroxyvitamin D 3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupolequadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease.

Liquid Chromatography-Tandem Mass Spectrometry Analysis of Acetaminophen Covalent Binding to Glutathione S-Transferases

“…Low resolving triple quadrupole systems (here a quadrupole-linear ion trap hybrid) generally reach lower limits of detection based on a higher duty cycle in optimized MRM modes. In comparison, quadrupole-time-of-flight instruments are less sensitive (Geib et al, 2016) but offer higher mass accuracy and the possibility for structural characterization by MS/MS. The difference in sensitivity is a crucial factor in characterizing low abundant protein modifications.…”

Absolute quantitation of acetaminophen‐modified human serum albumin in acute liver failure patients by liquid chromatography/tandem mass spectrometry

“…Validation was achieved by multiple assessments of custom-designed standards and QCs, in combination with linear agreement analysis by HRMS/MS, to investigate the presence of isobaric, serum-derived interferences. 35,36 The method yielded accuracies and %CVs well within the acceptable limits with fast sample analysis and clinical applicability. We focused on a simple protocol to follow, without the need for extensive fractionation.…”

“…This newly developed quantitative assay was successfully used to probe human acetaminophen covalent binding in vivo . Validation was achieved by multiple assessments of custom‐designed standards and QCs, in combination with linear agreement analysis by HRMS/MS, to investigate the presence of isobaric, serum‐derived interferences . The method yielded accuracies and %CVs well within the acceptable limits with fast sample analysis and clinical applicability.…”

MS1 ion current‐based quantitative proteomics: A promising solution for reliable analysis of large biological cohorts

“…Spectral counting (SpC) and other DDA‐MS2‐based methods (e.g., SpC‐Normalized Spectral Abundance Factor (NSAF) (Paoletti et al, ), Exponentially Modified Protein Abundance Index (emPAI) (Ishihama et al, ), MS2 Total‐Ion‐Current (TIC) (Tu et al, ), normalized Spectral Index (SIn) (Griffin et al, )), constitute a prevalently‐employed type of approach which measure a protein based on the total number of tandem mass spectra matching to peptides of the protein (Paoletti et al, ). The stochastic nature of DDA in precursor selection among different runs leads to considerable under‐sampling of low‐abundance, regulatory proteins (Zhou et al, ; Geib et al, ). Furthermore, dynamic exclusion which is devised to improve the depth of identification, substantially decreases the reproducibility of MS2 spectra acquisition among runs and thereby increasing the stochasticity and missing data of MS2‐based quantification.…”

“…Because of these issues, MS2‐based methods show low consistency in quantification, especially for low abundance ones; for example, as high as 20–50% identified proteins with missing quantitative values in 6–20 samples even higher when sample size increases (Bruderer et al, ; Zhang et al, ). To improve the reproducibility of protein measurement across a large number of biological replicates, the MS2‐based “data‐independent acquisition (DIA)” was developed, and the most prominent example is SWATH (sequential window acquisition of all theoretical fragment‐ion spectra), which triggers MS2 scans in a window‐based, independent and unbiased manner, and therefore alleviates the missing data to <10% at protein level for relatively large cohorts (Geib et al, ; Hu et al, Collins et al, ). Although MS2‐DIA represents an enormous advance in reproducible protein measurement, some limitations are also noted: (i) as DIA uses multiplexed fragmentation of many precursors, it may be difficult to interpret these MS2 spectra containing multiple co‐fragmented precursors while maintaining low false‐positives and (ii) the depth of identification using spectral library matching is often limited (Rost et al, ).…”