The use of tryptic marker‐peptides for the quantitative analysis of Cystatin C

Self Cite

A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cerebrospinal fluid (CSF). On column focusing on a C18 trapping column, in-line with the analytical column, was used for preconcentration. Quantification was performed with a triple quadrupole instrument in the multiple reaction monitoring mode. Weighted linear regression analysis proves to be a good linearity in a dynamic range of two orders of magnitude. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9914. Assay precision and accuracy were evaluated by direct injection of enkephalin fortified artificial CSF (aCSF) samples at three concentration levels. Mean accuracy of analysed concentrations was between 97.63 and 107.6%. LOD and LOQ were assessed at, respectively, 0.5 and 1 pmol/mL. Validation results show that it is feasible, with a capillary LC-MS/MS system, to quantify neuropeptides in the low femtomole range in aCSF. The obtained coefficients of variation, however, indicate that the use of appropriate isotopically labelled internal standards in neuropeptide quantification using narrow bore LC, combined with ESI-MS, may be highly beneficial.

Section: Resultsmentioning

confidence: 53%

Capillary liquid chromatography and tandem mass spectrometry for the quantification of enkephalins in cerebrospinal fluid

Sinnaeve

Storme

Bocxlaer

2005

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“…Independently, two QC samples of 37.5610 2 pmol/mL (50.0 lg/mL, QC 1 ) and 374 pmol/mL (5.00 lg/mL, QC 2 ) were also prepared. Marker peptides were generated using Promega Sequencing Grade Modified Trypsin (lyophilized powder, Leiden, The Netherlands) according to an earlier published method [11]. To that end, cystatin C is denatured for 1 h at 658C using a solution of 8 M guanidine -HCl, buffered to pH 8 (Sigma, Bornem, Belgium).…”

Section: Chemicalsmentioning

confidence: 99%

“…Additionally, the method was tested on cystatin C marker peptides, obtained after in-solution trypsinization according to a previously published method. Cystatin C is a 146 aminoacid (aa) protein of 13.3 kDa and was chosen as a model protein [11].…”

Section: Lc (Tandem) Ms (Lc-ms(/msmentioning

confidence: 99%

Improved analyte detectability of proteins and peptide lysates by means of multiple large‐volume injection in LC‐MS

Storme

t’Kindt

Bocxlaer³

2009

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A 'multiple (trapping) large-volume injection' approach was developed for the analysis of peptides and proteins. In this way, a maximally 10-fold gain in sensitivity could be achieved. The system involves the use of an automated 10-port switching valve in combination with a 1 mm i.d. trapping/guard column and a 1 mm i.d. x 150 mm analytical column. The optimized multiple injection/loading procedure allows quantitative measurements of peptides and protein lysates. Linear calibration curves (R(2) > or = 0.988) over a minimum of two orders of magnitude were generated for a range of peptide and protein standards with sensitivities equal to or even exceeding, those generally achieved only through increasing miniaturization (quantification limit > or = 0.5 pmol/mL).

“…Although more complex and less robust compared to the common immunochemical methods, it is less prone to cross-reactivity and has a competitive sensitivity. It has been demonstrated that several proteins like prions in brain tissue [7], the kidney dysfunction marker cystatin C [8], hemoglobin A2 in whole blood and dried blood spots [9], as well as the potential serum biomarker for prostate cancer Zn-a2 glycoprotein [10] can be quantitatively determined by this approach. Determination was achieved by first subjecting the sample to tryptic digestion and then subse-quently monitoring tryptic peptide(s) specific for the proteins to be determined.…”

Section: Introductionmentioning

confidence: 98%

Immuno‐capture as ultimate sample cleanup in LC‐MS/MS determination of the early stage biomarker ProGRP

Winther

Nordlund

Paus

et al. 2009

This paper presents a selective and efficient sample preparation procedure for NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker ProGRP, in human serum. The procedure is based on immuno-capture of ProGRP in 96-wells microtiter plates coated with the mAb E146. After immuno-capture and thorough rinse, trypsin was added for in-well digestion. Subsequently the signature peptide was enriched by SPE and determined by LC-MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole MS was used during optimization of the method. A triple quadropole MS was used in the method evaluation in order to improve sensitivity. The evaluation showed good repeatability (RSD, 11.9-17.5%), accuracy (3.0-6.6%), and linearity (r(2) = 0.995) in the tested range (0.5-50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and 0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was compared to a previous published LC-MS method for the determination of ProGRP in serum based on protein precipitation and online sample cleanup. Both showed acceptable method performance, however, the immuno-capture LC-MS method was superior with respect to sensitivity. This illustrates the large potential of immuno-capture sample preparation prior to LC-MS in protein biomarker quantification.