An LC-MS/MS-based platform for the quantification of multiple amyloid beta peptides in surrogate cerebrospinal fluid.

Introduction: The accurate quantification of amyloid beta (Aß) peptides in cerebrospinal fluid (CSF) is crucial for Alzheimer's disease (AD) research, particularly in terms of preclinical and biomarker studies. Traditional methods, such as the enzyme-linked immunosorbent assay (ELISA), have limitations. These include high costs, labor intensity, lengthy processes, and the possibility of cross-reactivity. Objectives: The primary objectives of this research were twofold: to comprehensively characterize Aß peptides and to develop a reliable and accurate method for the simultaneous quantification of Aß 1-40 and Aß 1-42 peptides in surrogate CSF that is traceable to the International System of Units (SI). Methods: We developed a novel method that combined solid phase extraction (SPE) with isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC/MSMS). SPE was employed to efficiently eliminate matrix interferences, while [15N] Aß1-40 and [15N] Aß1-42 served as internal standards to improve accuracy. In addition, we introduced Peptide Impurity Corrected Amino Acid Analysis (PICAA) to ensure traceability to the SI and reliable quantification of Aß peptides. Results: The developed platform demonstrated a linear calibration range of 300-20000 pg/ml for both Aß1-42 and Aß1-40 peptides, accompanied by strong correlation coefficients greater than 0.995. Quality Control (QC) samples demonstrated an accuracy of at least 90.0 %. Conclusion: The enhanced specificity and flexibility of the developed platform potentially have implications for Alzheimer's disease diagnosis and future investigations of novel Aß peptide biomarkers.

Development and optimisation of a generic micro LC-ESI-MS method for the qualitative and quantitative determination of 30-mer toxic gliadin peptides in wheat flour for food analysis.

We sometimes see manufactured bakery products on the market which are labelled as being gluten free. Why is the content of such gluten proteins of importance for the fabrication of bakery industry and for the products? The gluten proteins represent up to 80 % of wheat proteins, and they are conventionally subdivided into gliadins and glutenins. Gliadins belong to the proline and glutamine-rich prolamin family. Its role in human gluten intolerance, as a consequence of its harmful effects, is well documented in the scientific literature. The only known therapy so far is a gluten-free diet, and hence, it is important to develop robust and reliable analytical methods to quantitatively assess the presence of the identified peptides causing the so-called coeliac disease. This work describes the development of a new, fast and robust micro ion pair-LC-MS analytical method for the qualitative and quantitative determination of 30-mer toxic gliadin peptides in wheat flour. The use of RapiGest™ SF as a denaturation reagent prior to the enzymatic digestion showed to shorten the measuring time. During the optimisation of the enzymatic digestion step, the best 30-mer toxic peptide was identified from the maximum recovery after 3 h of digestion time. The lower limit of quantification was determined to be 0.25 ng/µL. The method has shown to be linear for the selected concentration range of 0.25-3.0 ng/µL. The uncertainty related to reproducibility of measurement procedure, excluding the extraction step, has shown to be 5.0 % (N = 12). Finally, this method was successfully applied to the quantification of 30-mer toxic peptides from commercial wheat flour with an overall uncertainty under reproducibility conditions of 6.4 % including the extraction of the gliadin fraction. The results were always expressed as the average of the values from all standard concentrations. Subsequently, the final concentration of the 30-mer toxic peptide in the flour was calculated and expressed in milligrams per gram unit. The determined, calculated concentration of the 30-mer toxic peptide in the flour was found to be 1.29 ± 0.37 µg/g in flour (N = 25, s y = 545,075, f = 25 - 2 (t = 2.069), P = 95 %, two-sided).

Development and validation of a generic method for quantification of collagen in food supplement tablets using liquid chromatography coupled with time-of-flight mass spectrometry.

A generic method for the quantification of type II collagen in protein-based dietary supplements is described. This quantitative analysis was conducted using liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (LC-ESI-TOF MS). Compared to classical methods with the use of isotope-labeled standards, our method includes, for the first time, the quantification of hydroxyproline using histidine as an internal standard. Separation of the analytes was performed on a Phenomenex Synergi 4 µm Fusion-RP 80 Ǻ column (150 × 2.0 mm, 4.0 µm) with a mobile phase made of 10 mM ammonium formate in water (A) and 10 mM ammonium formate in methanol (B). The assay was fully validated according to FDA guidelines, and the method exhibited sufficient specificity, accuracy, and precision. Intra- and inter-batch accuracy, determined as a deviation between nominal and measured values, ranged from -4.8 to 9.1% and from 0.9 to 6.4 %, respectively. All analytes (hydroxyproline and histidine) at three concentration levels showed extraction recoveries from 89 to 98 %. The method was successfully applied to protein-based dietary supplements of the pharmaceutical industry.

Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.

A generic LC-MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1-34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC-MS setup, large volumes of filtrates (e.g., 2×750 µL) were injected and the peptides were trapped on a 1mm i.d.×10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments.

Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

Kinetic study of neuropeptide Y (NPY) proteolysis in blood and identification of NPY3-35: a new peptide generated by plasma kallikrein.

There is little information on how neuropeptide Y (NPY) proteolysis by peptidases occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive forms and also because the factors affecting the expression of these enzymes have been poorly studied. In the present study, LC-MS/MS was used to identify and quantify NPY fragments resulting from peptidolytic cleavage of NPY(1-36) upon incubation with human serum. Kinetic studies indicated that NPY(1-36) is rapidly cleaved in serum into 3 main fragments with the following order of efficacy: NPY(3-36) >> NPY(3-35) > NPY(2-36). Trace amounts of additional NPY forms were identified by accurate mass spectrometry. Specific inhibitors of dipeptidyl peptidase IV, kallikrein, and aminopeptidase P prevented the production of NPY(3-36), NPY(3-35), and NPY(2-36), respectively. Plasma kallikrein at physiological concentrations converted NPY(3-36) into NPY(3-35). Receptor binding assays revealed that NPY(3-35) is unable to bind to NPY Y1, Y2, and Y5 receptors; thus NPY(3-35) may represent the major metabolic clearance product of the Y2/Y5 agonist, NPY(3-36).