Quantification of hepcidin using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Hepcidin is known to be a key systemic iron‐regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation‐exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI‐TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70–80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within ‐day repeatability (CV <13%) and between‐day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Study of human neutrophil peptides in saliva by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry is used to rapidly characterize the human neutrophil peptides – HNP 1, 2, and 3 – in saliva. The saliva excreted from the parotid and sublingual/submandibular glands of 70 individuals were collected and examined using MALDI‐TOF. The MALDI approach requires no sample pretreatment other than mixing the saliva‐absorbing material with the matrix and drying under ambient conditions. Tissue paper was the best material for collecting the saliva samples because of its strong texture and high absorbance, and sinapinic acid was the best MALDI matrix for the analysis of the HNPs. HNPs were detected in almost all the samples collected from the parotid glands, with no obvious differences among age or gender. In contrast, the distribution of the HNPs in the samples collected from the sublingual/submandibular glands was age‐dependent: no HNPs were detected for those collected from individuals younger than 30, but the HNPs were present in all of the samples collected from those older than 60 years. The increased probability of detecting saliva HNPs with age suggests that HNPs may function as a biomarker for aging. Copyright © 2009 John Wiley & Sons, Ltd.     

Negative ion production from peptides and proteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Negative ion production from peptides and proteins was investigated by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Although most research on peptide and protein identification with ionization by MALDI has involved the detection of positive ions, for some acidic peptides protonated molecules are not easily formed because the side chains of acidic residues are more likely to lose a proton and form a deprotonated species. After investigating more than 30 peptides and proteins in both positive and negative ion modes, [M–H]⁻ ions were detected in the negative ion mode for all peptides and proteins although the matrix used was 2,5‐dihydroxybenzoic acid (DHB), which is a good proton donor and favors the positive ion mode production of [M+H]⁺ ions. Even for highly basic peptides without an acidic site, such as myosin kinase inhibiting peptide and substance P, good negative ion signals were observed. Conversely, gastrin I (1‐14), a peptide without a highly basic site, will form positive ions. In addition, spectra obtained in the negative ion mode are usually cleaner due to absence of alkali metal adducts. This can be useful during precursor ion isolation for MS/MS studies. Copyright © 2008 John Wiley & Sons, Ltd.     

Detection of pathogenic Verticillium spp. using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Verticillium spp. have been listed by the European and Mediterranean Plant Protection Organization (EPPO) and China as plant quarantine pests. Although attempts have been made to develop a simple routine laboratory assay to detect these organisms, none are routinely used. We describe for the first time a robust assay for reliable identification of Verticillium spp. using protein fingerprinting data obtained by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry(MALDI‐TOF‐MS). Several sample preparation methods and matrices were investigated to improve mass spectra for the routine identification of six species of Verticillium spp.(Verticillium dahiliae, V. alboatrum, V. fungicola, V. nigrescens, and V. lecanii) by MALDI‐TOF‐MS. Using the optimized experimental method, we constructed a protein fingerprint database for six species of Verticillium and established a analysis criteria of log(Score). This MALDI‐TOF‐MS protocol should prove useful as a rapid and reliable assay for distinguishing different Verticillium spp. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry for the study of Helicobacter pylori

The characteristics of matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry based investigation of extremely variable bacteria such as Helicobacter pylori were studied. H. pylori possesses a very high natural variability. Accurate tools for species identification and epidemiological characterization could help the scientific community to better understand the transmission pathways and virulence mechanisms of these bacteria. Seventeen clinical as well as two laboratory strains of H. pylori were analyzed by the MALDI Biotyper method for rapid species identification. Mass spectra collected were found containing 7–13 significant peaks per sample, and only six protein signals were identical for more than half of the strains. Four of them could be assigned to ribosomal proteins RL32, RL33, RL34, and RL36. The reproducible peak with m/z 6948 was identified as a histidine‐rich metal‐binding polypeptide by tandem mass spectrometry (MS/MS). In spite of the evident protein heterogeneity of H. pylori the mass spectra collected for a particular strain under several cultivations were highly reproducible. Moreover, all clinical strains were perfectly identified as H. pylori species through comparative analysis using the MALDI Biotyper software (Bruker Daltonics, Germany) by pattern matching against a database containing mass spectra from different microbial strains (n = 3287) including H. pylori 26695 and J99. The results of this study allow the conclusion that the MALDI‐TOF direct bacterial profiling is suited for H. pylori identification and could be supported by mass spectra fragmentation of the observed polypeptide if necessary. Copyright © 2010 John Wiley & Sons, Ltd.     

Aquatic fulvic acid as a matrix for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis

Aquatic fulvic acids (AFAs) are demonstrated to be effective matrices for the analysis of various polar compounds (ranging from 100–1500 Da) by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS). The efficiency of AFA as a matrix is shown for a wide range of test compounds, including a number of carbohydrates, cyclodextrins and peptides, with typical detection limits of ～10 µg mL^?1. The propensity of AFA to enhance ionization through protonation of peptides, and formation of sodium and potassium adducts of carbohydrates and polyethylene glycol, was noted. Differences were observed in the performances of the two AFA matrices used, a Suwannee River, International Humic Substances Society (IHSS) standard and a locally extracted fulvic acid (LFA). For example, in the analysis of carbohydrate standards, the use of the LFA matrix typically doubled the analyte ion signal intensities and resulted in signal‐to‐noise (S/N) ratios that were 2–4 times better than when the Suwannee River AFA matrix was used. AFA was also used in the analysis of real‐world samples without extraction or purification; cantaloupe juice and acetaminophen tablets were analyzed, and glucose and acetaminophen could easily be identified as respective components. When lower concentrations of fulvic acid were used in the presence of sugars, a reversal of roles was observed in which the sugars functioned as the matrix and significantly enhanced ionization of the AFA components, while ions associated with the sugars themselves were suppressed or absent. Effective as a matrix for a variety of analytes and widely available, AFA is an attractive environmentally friendly choice for use in MALDI applications. Copyright © 2006 John Wiley & Sons, Ltd.     

Species identification by analysis of bone collagen using matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry

Species identification of fragmentary bone, such as in rendered meat and bone meal or from archaeological sites, is often difficult in the absence of clear morphological markers. Here we present a robust method of analysing genus‐specific collagen peptides by mass spectrometry simply by using solid‐phase extraction (a C18 ZipTip®) for peptide purification, rather than liquid chromatography/mass spectrometry (LC/MS). Analysis of the collagen from 32 different mammal species identified a total of 92 peptide markers that could be used for species identification, for example, in processed food and animal feed. A set of ancient (>100 ka@10°C) bone samples was also analysed to show that the proposed method has applications to archaeological bone identification. Copyright © 2009 John Wiley & Sons, Ltd.     

Matrix/analyte ratio influencing polymer molecular weight distribution in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) has been used to characterize poly(L‐lysine) polymers and unique oligomer peptides, like 10‐, 15‐ and 25‐mer [Lys]_n oligolysine peptides. Several matrices have been tried in order to find optimal conditions, but only α‐cyano‐4‐hydroxycinnamic acid gave analytically useful spectra. The synthetic oligomers and their mixtures gave good quality spectra, showing protonated and cationized molecules, including doubly charged species. The polymers, analogously, gave a wide distribution of single‐ and double‐cationized peak series. The polymer distributions observed indicate the presence of significant suppression effects. The concentration (matrix/analyte ratio) was found to influence the results significantly; distributions shifting to higher masses when higher polymer concentrations were used. This effect was studied in detail using the synthetic (‘monodisperse’) oligolysine peptides. It was found that the relative intensities change by over an order of magnitude in the 0.1–10 pmol/µL concentration range (typical for most proteomic analyses). The results indicate that concentration effects need to be considered when MALDI‐MS is used for quantitative purposes. Copyright © 2009 John Wiley & Sons, Ltd.