Calibration of mass spectrometric peptide mass fingerprint data without specific external or internal calibrants

BACKGROUND: Peptide Mass Fingerprinting (PMF) is a widely used mass spectrometry (MS) method of analysis of proteins and peptides. It relies on the comparison between experimentally determined and theoretical mass spectra. The PMF process requires calibration, usually performed with external or internal calibrants of known molecular masses. RESULTS: We have introduced two novel MS calibration methods. The first method utilises the local similarity of peptide maps generated after separation of complex protein samples by two-dimensional gel electrophoresis. It computes a multiple peak-list alignment of the data set using a modified Minimum Spanning Tree (MST) algorithm. The second method exploits the idea that hundreds of MS samples are measured in parallel on one sample support. It improves the calibration coefficients by applying a two-dimensional Thin Plate Splines (TPS) smoothing algorithm. We studied the novel calibration methods utilising data generated by three different MALDI-TOF-MS instruments. We demonstrate that a PMF data set can be calibrated without resorting to external or relying on widely occurring internal calibrants. The methods developed here were implemented in R and are part of the BioConductor package mscalib available from . CONCLUSION: The MST calibration algorithm is well suited to calibrate MS spectra of protein samples resulting from two-dimensional gel electrophoretic separation. The TPS based calibration algorithm might be used to correct systematic mass measurement errors observed for large MS sample supports. As compared to other methods, our combined MS spectra calibration strategy increases the peptide/protein identification rate by an additional 5 – 15%

Analytical model of peptide mass cluster centres with applications

BACKGROUND: The elemental composition of peptides results in formation of distinct, equidistantly spaced clusters across the mass range. The property of peptide mass clustering is used to calibrate peptide mass lists, to identify and remove non-peptide peaks and for data reduction. RESULTS: We developed an analytical model of the peptide mass cluster centres. Inputs to the model included, the amino acid frequencies in the sequence database, the average length of the proteins in the database, the cleavage specificity of the proteolytic enzyme used and the cleavage probability. We examined the accuracy of our model by comparing it with the model based on an in silico sequence database digest. To identify the crucial parameters we analysed how the cluster centre location depends on the inputs. The distance to the nearest cluster was used to calibrate mass spectrometric peptide peak-lists and to identify non-peptide peaks. CONCLUSION: The model introduced here enables us to predict the location of the peptide mass cluster centres. It explains how the location of the cluster centres depends on the input parameters. Fast and efficient calibration and filtering of non-peptide peaks is achieved by a distance measure suggested by Wool and Smilansky

MASS SPECTROMETRIC STUDIES OF NOVEL LIPID-BASED NANOMATERIALS: INVESTIGATIONS OF THE INTRACELLULAR FATE

Dicatonic gemini surfactants have, within the past two decades, demonstrated advancing non-viral gene transfection ability in cell cultures and in animal models. However, knowledge of the intracellular/subcellular fate of gemini surfactants that may further advance gemini surfactant-based gene transfection is very limited. Therefore, my Ph.D. research conducted the investigation of gemini surfactants within transfected PAM212 keratinocytes including the development of effective bioanalytical mass spectrometric (MS) methods necessary for such investigations. For effective mass spectrometric bioanalysis of the gemini surfactants within cellular matrix their fingerprint fragment ions necessary for targeted identification and quantification were first determined through single-stage (MS), tandem (MS/MS) and multi-stage (MS3) analyses. The molecular composition of gemini surfactants was confirmed. In addition, fragmentation mechanisms of novel dipyridinium and β-cyclodextrin-based diquaternary ammonium molecules (chosen as study compounds) were established in detail, allowing for their qualititive and quantitative analysis. Hydrophilic interaction liquid chromatography-based (HILIC)-MS/MS methods, alone and in conjunction with the method of standard addition, were subsequently developed/validated by adopting multiple reaction monitoring (MRM), ensuring selectivity alongside the distinctive chromatographic separation. The analytical strategy ensured selectivity/specificity for target gemini surfactants including two lead compounds, 16(Py)-S-2-S-(Py)16 and 16-3-16. The validated HILIC-MS/MS methods were more sensitive, faster and have simplified isocratic elution relative to recently reported methods. In the application to nanoparticle-based gene transfection studies, the HILIC-MS/MS and standard addition–HILIC-MS/MS methods allowed a comprehensive investigation of the cellular uptake, intracellular deposition and subcellular distribution of the 16(Py)-S-2-S-(Py)16 and 16-3-16 gemini surfactants. The results showed similar cellular uptake and intracellular depletion trends but different subcellular distribution profiles. Both gemini surfactants showed an initial spike in their concentration within cells upon addition of gemini surfactant-based DNA nanoparticles to the cells, as would be expected to achieve nanoparticle entry into cells during transfection. The intracellular gemini surfactant content, however, underwent a depletion upon removal of the added nanoparticles – a trend observed for the first time and attributable to either gemini surfactant biodegradation or exocytosis from host cells. Results of subcellular distribution showed higher distribution of 16-3-16 to the mitochondria and nucleus relative to its 16(Py)-S-2-S-(Py)16 counterpart (p < 0.05), with the two having similar distribution to the cell membrane, cytosol and the remnant subcellular residue. This differential subcellular distribution, determined for the first time, may explain a suggested higher toxicity for 16-3-16 as its increased distribution to the mitochondria and nucleus could impact their biological integrity and function. Herein, the investigations and findings will benefit further exploration of gemini surfactants through the established molecular fragmentation fingerprints of novel compounds and comprehensive LC-MRM-MS bioanalytical strategies for studying the biological fate, elucidating varying toxicity and assessing possible metabolite formation

Development of quantitative structure property relationships to support non-target LC-HRMS screening

Κατά την τελευταία δεκαετία, ένας μεγάλος αριθμός αναδυόμενων ρύπων έχουν ανιχνευθεί και ταυτοποιηθεί σε επιφανειακά ύδατα και λύματα, προκαλώντας ανησυχία για το υδάτινο οικοσύστημα, λόγω της πιθανής χημικής τους σταθερότητας. Η τεχνική της υγροχρωματογραφίας - φασματομετρίας μάζας υψηλής διακριτικής ικανότητας (LC-HRMS) αποτελεί μια αποτελεσματική τεχνική για την ανίχνευση αναδυόμενων ρύπων στο περιβάλλον. Η ταυτόχρονη δε ανάλυση των δειγμάτων με τις συμπληρωματικές τεχνικές της υγροχρωματογραφίας αντίστροφης φάσης (RPLC) και της υγροχρωματογραφίας υδρόφιλων αλληλεπιδράσεων (HILIC), συντελεί στην ταυτοποίηση «ύποπτων» ή και άγνωστων ρύπων με ποικίλες φυσικοχημικές ιδιότητες. Για την ταυτοποίηση τους, απαιτείται να πληρούνται συγκεκριμένα κριτήρια, τα οποία αξιολογούνται με βάση τη χρήση διαγνωστικών εργαλείων, όπως η ακριβής πρόβλεψη του χρόνου ανάσχεσης, η in silico θραυσματοποίηση και η πρόβλεψη της συμπεριφορά τους στον ιοντισμό. Στο 3ο κεφάλαιο της παρούσας διδακτορικής διατριβής περιγράφεται η ανάπτυξη μιας ολοκληρωμένης πορείας εργασίας (workflow) για τη διερεύνηση των παραμέτρων που επηρεάζουν τον χρόνο έκλουσης μεγάλου αριθμού ενώσεων που συγκαταλέγονται στους αναδυόμενους ρύπους. Για τον σκοπό αυτό, πάνω από 2.500 αναδυόμενοι ρύποι χρησιμοποιήθηκαν για την ανάπτυξη του μοντέλου πρόβλεψης χρόνου ανάσχεσης για τις 2 υγροχρωματογραφικές τεχνικές (RP- και HILIC-LC-HRMS) και για ηλεκτροψεκασμό τόσο σε θετικό όσο και σε αρνητικό ιοντισμό (+/-ESI). Στη συνέχεια, πραγματοποιήθηκε εφαρμογή του μοντέλου για την υπολογιστική πρόβλεψη του χρόνου ανάσχεσης, για την ταυτοποίηση 10 νέων προϊόντων μετασχματισμού των φαρμακευτικών ενώσεων (tramadol, furosemide και niflumic acid) ύστερα από επεξεργασία με όζον. Στο 4ο κεφάλαιο παρουσιάζεται η ανάπτυξη ενός καινοτόμου γενικευμένου χημειομετρικού μοντέλου το οποίο είναι ικανό να προβλέπει τον χρόνο έκλουσης κάθε πιθανού ρύπου, ανεξαρτήτου υγροχρωματογραφικής μεθόδου που χρησιμοποιείται, συμβάλλοντας σημαντικά στην σύγκριση αποτελεσμάτων από διαφορετικές LC-HRMS μεθόδους. Το συγκεκριμένο μοντέλο χρησιμοποιήθηκε για την ταυτοποίηση «ύποπτων» και άγνωστων ενώσεων σε διεργαστηριακές δοκιμές. Το Κεφάλαιο 5, περιέχει την περιγραφή της ανάπτυξης ενός υπολογιστικού μοντέλου πρόβλεψης τοξικότητας αναδυόμενων ρύπων που ανιχνεύονται στο υδάτινο οικοσύστημα. Το συγκεκριμένο μοντέλο αποσκοπεί στην εκτίμηση του πιθανού περιβαλλοντικού κινδύνου για νέες ενώσεις που ταυτοποιήθηκαν μέσω σάρωσης «ύποπτων» ενώσεων και μη-στοχευμένης σάρωσης, για τις οποίες δεν είναι ακόμα διαθέσιμα πειραματικά δεδομένα τοξικότητας. Τέλος, στο κεφάλαιο 6 παρουσιάζεται ένας αυτοματοποιημένος και συστηματικός τρόπος σάρωσης «ύποπτων» ενώσεων και μη-στοχευμένης σάρωσης σε δεδομένα από LC-HRMS. Η νέα αυτή αυτοματοποιημένη πορεία εργασίας, αποσκοπεί στην λιγότερο χρονοβόρα επεξεργασία των HRMS δεδομένων, και στην εφαρμογή της μη-στοχευμένης σάρωσης ώστε να είναι δυνατή η εφαρμογή τους σε καθημερινούς ελέγχους ρουτίνας ή/και για χρήση από τις κανονιστικές αρχές.Over the last decade, a high number of emerging contaminants were detected and identified in surface and waste waters that could threaten the aquatic environment due to their pseudo-persistence. As it is described in chapters 1 and 2, liquid chromatography high resolution mass spectroscopy (LC-HRMS) can be used as an efficient tool for their screening. Simultaneously screening of these samples by hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) would help with full identification of suspects and unknown compounds. However, to confirm the identity of the most relevant suspect or unknown compounds, their chemical properties such as retention time behavior, MSn fragmentation and ionization modes should be investigated. Chapter 3 of this thesis discusses the development of a comprehensive workflow to study the retention time behavior of large groups of compounds belonging to emerging contaminants. A dataset consisted of more than 2500 compounds was used for RP/HILIC-LC-HRMS, and their retention times were derived in both Electrospray Ionization mode (+/-ESI). These in silico approaches were then applied on the identification of 10 new transformation products of tramadol, furosemide and niflumic acid (under ozonation treatment). Chapter 4 discusses about the development of a first retention time index system for LC-HRMS. Some practical applications of this RTI system in suspect and non-target screening in collaborative trials have been presented as well. Chapter 5 describes the development of in silico based toxicity models to estimate the acute toxicity of emerging pollutants in the aquatic environment. This would help link the suspect/non-target screening results to the tentative environmental risk by predicting the toxicity of newly tentatively identified compounds. Chapter 6 introduces an automatic and systematic way to perform suspect and non-target screening in LC-HRMS data. This would save time and the data analysis loads and enable the routine application of non-target screening for regulatory or monitoring purpose

Analysis of the effects of BrdU on DLKP human lung cancer cells by two-dimensional difference gel electrophoresis and mass spectrometry

Bromodeoxyuridine (BrdU) is a thymidiie analogue that incorporates into DNA of dividing cells during the S-phase of the cell cycle. Previous work in laboratories reported that treatment with lOyM BrdU in the human lung carcinoma cell line (DLKP) resulted in increased expression of the cytoskeletal proteins Keratin 8 and 18 and the cell adhesion proteins a2 and b1 integrin. This study investigated protein expression changes in differentiating DLKP cells following exposure to 10yM BrdU. DLKP cells were grown in culture flasks and harvested after 7 days exposure to BrdU. Two-dimensional gel electrophoresis was used to investigate BrdU specific changes in the proteome of DLKP BrdU treated and control cells. Cy3-labeled DLKP control were combined with Cy-5 labeled BrdU DLKP treated proteins and separated on the same 2-D gel along with a Cy-2 labelled mixture of both samples as an internal standard. Using DIGE technology, the statistically significant comparisons of each protein abundance was made over three biological replicates. 43 protein spots were identified as differentially regulated. Among the 43 protein spots, 25 were found to be up-regulated and 18 were found to be downregulated

Molecular, mass spectral, and physiological analyses of orcokinins and orcokinin precursor-related peptides in the lobster Homarus americanus and the crayfish Procambarus clarkii

Recently, cDNAs encoding prepro-orcokinins were cloned from the crayfish Procambarus clarkii; these cDNAs encode multiple copies of four orcokinin isoforms as well as several other peptides. Using the translated open reading frames of the P. clarkii transcripts as queries, five ESTs encoding American lobster Homarus americanus orthologs were identified via BLAST analysis. From these clones, three cDNAs, each encoding one of two distinct prepro-hormones, were characterized. Predicted processing of the deduced prepro-hormones would generate 13 peptides, 12 of which are conserved between the 2 precursors: the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), FDAFTTGFGHN (an orcomyotropin-related peptide), SSEDMDRLGFGFN, GDY(SO3)DVYPE, VYGPRDIANLY and SAE. Additionally, one of two longer peptides (GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV or APARSSPQQDAAAGYTDGAPV) is predicted from each prepro-hormone. MALDI-FTMS analyses confirmed the presence of all predicted orcokinins, the orcomyotropin-related peptide, and three precursor-related peptides, SSEDMDRLGFGFN, GDYDVYPE (unsulfated) and VYGPRDIANLY, in H. americanus neural tissues. SAE and the longer, unshared peptides were not detected. Similar complements of peptides are predicted from P. clarkii transcripts; the majority of these were detected in its neural tissues with mass spectrometry. Truncated orcokinins not predicted from any precursor were also detected in both species. Consistent with previous studies in the crayfish Orconectes limosus, NFDEIDRSGFGFN increased mid-/hindgut motility in P. clarkii. Surprisingly, the same peptide, although native to H. americanus, did not affect gut motility in this species. Together, our results provide the framework for future investigations of the regulation and physiological function of orcokinins/orcokinin precursor-related peptides in astacideans. © 2008 Elsevier Inc. All rights reserved

A method for improving SELDI-TOF mass spectrometry data quality

<p>Abstract</p> <p>Background</p> <p>Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a powerful tool for rapidly generating high-throughput protein profiles from a large number of samples. However, the events that occur between the first and last sample run are likely to introduce technical variation in the results.</p> <p>Methods</p> <p>We fractionated and analyzed quality control and investigational serum samples on 3 Protein Chips and used statistical methods to identify poor-quality spectra and to identify and reduce technical variation.</p> <p>Results</p> <p>Using diagnostic plots, we were able to visually depict all spectra and to identify and remove those that were of poor quality. We detected a technical variation associated with when the samples were run (referred to as batch effect) and corrected for this variation using analysis of variance. These corrections increased the number of peaks that were reproducibly detected.</p> <p>Conclusion</p> <p>By removing poor-quality, outlier spectra, we were able to increase peak detection, and by reducing the variance introduced when samples are processed and analyzed in batches, we were able to increase the reproducibility of peak detection.</p

The development of high-throughput mass spectrometric methods for the qualitative and quantitative analysis of diquaternary ammonium gemini surfactants

For over a decade, diquaternary ammonium gemini surfactants have shown promise as non-viral gene delivery agents in both in vitro and in vivo systems. Their continued development, however, requires an understanding of their biological fate. The absence of identification and quantification methods that can achieve that goal is what drove the development of simple and rapid mass spectrometry (MS)-based methods; the focus of my Ph.D. dissertation. Prior to the development of these MS-based methods, an understanding of the gas phase behavior of diquaternary ammonium gemini surfactants is required. The development of a universal fragmentation pathway for gemini surfactants was achieved using low resolution and high resolution MS instruments. Single stage (MS), tandem stage (MS/MS and quasi-multi-stage (quasi MS3) mass spectrometry analysis allowed for the confirmation of the molecular composition and structure of each gemini surfactant through the identification of common and unique mass to charge values. Understanding the fragmentation behavior allowed for the specific identification and/or quantification of gemini surfactants by MS-based methods; including liquid chromatography low resolution tandem mass spectrometry (LC-LR-MS/MS), fast chromatography low resolution tandem mass spectrometry, fast chromatography high resolution mass spectrometry, desorption electrospray ionization low resolution mass spectrometry and matrix assisted laser desorption ionization high resolution mass spectrometry. We hypothesized that a LC-LR-MS/MS method would be the most effective quantitative method for the quantification of N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3) within PAM212 cellular lysate; achieving the lowest lower limit of quantification (LLOQ). Although the LC-LR-MS/MS method achieved a LLOQ suitable for analysis of G16-3 within PAM212 cell lysate, its limitations made it an inefficient method. In comparison, the four alternative mass spectrometry methods were faster, more efficient and less expensive than a conventional LC-LR-MS/MS method for the post transfection quantification of G16-3 within PAM212 cell lysate to be determined; 1.45 ± 0.06 μM. Future application of the universal fragmentation pathway and each MS-based quantification method will be beneficial for the future development of diquaternary ammonium gemini surfactants to further understand their post transfection fate

Prediction of collision cross section values: application to non-intentionally added substance identification in food contact materials

The synthetic chemicals in food contact materials can migrate into food and endanger human health. In this study, the traveling wave collision cross section in nitrogen values of more than 400 chemicals in food contact materials were experimentally derived by traveling wave ion mobility spectrometry. A support vector machine-based collision cross section (CCS) prediction model was developed based on CCS values of food contact chemicals and a series of molecular descriptors. More than 92% of protonated and 81% of sodiated adducts showed a relative deviation below 5%. Median relative errors for protonated and sodiated molecules were 1.50 and 1.82%, respectively. The model was then applied to the structural annotation of oligomers migrating from polyamide adhesives. The identification confidence of 11 oligomers was improved by the direct comparison of the experimental data with the predicted CCS values. Finally, the challenges and opportunities of current machine-learning models on CCS prediction were also discussed. © 2022 The Authors. Published by American Chemical Society