Development of high-sensitivity atmospheric pressure (ap) matrix-assisted laser desorption/ionization (maldi) and open air ionization techniques for the analysis of biomolecules by mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) has been celebrated as a soft ionization method for analyzing very diverse biological species including large proteins, peptides, carbohydrates, lipids and metabolites. The fact that MALDI is tolerant to salts and buffers and that it mostly produces singly charged ions from intact biomolecules is considered highly advantageous over electrospray ionization (ESI). Almost two decades after the introduction of vacuum MALDI, the technique was successfully implemented under atmospheric pressure (AP) conditions by Laiko and co-workers. Some of the most salient advantages of AP-MALDI over vacuum MALDI are its ability to generate intact ions from labile species with minimal fragmentation due to collisional cooling under AP, the ability of performing MSn experiments, and its exchangeability with other ion sources. However, AP-MALDI suffers from limited sensitivity due to low ion transmission efficiency under AP conditions. Because sensitivity is a function of the sample pretreatment method of choice, both preconcentration and selective sample fractionation can be used during the initial stages of the analytical pipeline to improve detectability. To that end, the first part of the work presented in this thesis is aimed at investigating various approaches to improve the sensitivity of AP-MALDI for mass spectrometric analysis of biomolecules. Chapter 1 reviews the history of laser desorption ionization (LDI), presenting salient features of vacuum MALDI and AP-MALDI, and concludes with a brief overview of leading ambient ionization techniques, such as Direct Analysis in Real Time (DART) ionization. Chapter 2 presents an investigation of an on chip sample preconcentration approach coupled to AP-MALDI for high-sensitivity analysis of neuropeptides extracted from Aedes aegypti mosquito heads. The theme of exploring efficient and reproducible purification methods for complex biosamples is continued in Chapter 3, where an evaluation of new on-tip solid-phase extraction (SPE) micro columns with various functional groups is presented. A second approach for enhancing AP-MALDI sensitivity by constructing a new pneumatically-assisted (PA) AP-MALDI ion source is presented in Chapter 4, where various factors affecting the performance of this device are investigated. Chapter 5 describes work involving the evaluation of DART ionization as a high-throughput method for the detection and identification of small terpene molecules central to the Aedes aegypti mosquito lifecycle.Ph.D.Committee Chair: Fernandez, Facundo M.; Committee Member: Brown, Ken R; Committee Member: Merrill, Alfred H. Jr; Committee Member: Noriega, Fernando G.; Committee Member: Orlando, Thomas M

Serum biomarker profiling by solid‐phase extraction with particle‐embedded micro tips and matrix‐assisted laser desorption/ionization mass spectrometry

One of the main challenges in high-throughput serum profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the development of proteome fractionation approaches that allow the acquisition of reproducible profiles with a maximum number of spectral features and minimum interferences from biological matrices. This study evaluates a new class of solid-phase extraction (SPE) pipette tips embedded with different chromatographic media for fractionation of model protein digests and serum samples. The materials embedded include strong anion exchange (SAX), weak cation exchange (WCX), C18, C8, C4, immobilized metal affinity chromatography (IMAC) and zirconium dioxide particles. Simple and rapid serum proteome profiling protocols based on these SPE micro tips are described and tested using a variety of MALDI matrices. We show that different types of particle-embedded SPE micro tips provide complementary information in terms of the spectral features detected for β-casein digests and control human serum samples. The effect of different sample pretreatments, such as serum dilution and ultrafiltration using molecular weight cut-off membranes, and the reproducibility observed for replicate experiments, are also evaluated. The results demonstrate the usefulness of these simple SPE tips combined with offline MALDI-TOF MS for obtaining information-rich serum profiles, resulting in a robust, versatile and reproducible open-source platform for serum biomarker discovery

Rapid direct analysis in real time (DART) mass spectrometric detection of juvenile hormone III and its terpene precursors.

Direct analysis in real time (DART) is a plasma-based ambient ionization technique that enables rapid ionization of small molecules with high sample throughput. In this work, DART was coupled to an orthogonal (oa) time-of-flight (TOF) mass spectrometer and the system was optimized for analyzing a vital hormonal regulator in insects, juvenile hormone (JH) III and its terpene precursors, namely, farnesol, farnesoic acid, and methyl farne-soate. Optimization experiments were planned using design of experiments (DOE) full factorial models to identify the most significant DART variables contributing to JH III analysis sensitivity by DART-TOF mass spectrometry (MS). The optimized DART-TOF MS method had femto-mole to sub-picomole detection limits for terpene standards, along with mass accuracies below 5 ppm. Finally, the possibility of distinguishing between two farnesol isomers by in-source-collision-induced dissociation (CID) in the first differentially pumped region of the oaTOF mass spectrometer was investigated. DART-MS enabled high-throughput, sensitive analysis with acquisition times ranging from 30 s to a minute. To the best of our knowledge, this is the first report on the application of DART-MS to the detection and identification of volatile or semi-volatile insect terpenoids, and on the use of DOE approaches to optimize DART-MS analytical procedures

On-chip solid-phase extraction pre-concentration/ focusing substrates coupled to atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric analysis of biomolecules. AP-MALDI and electrospray ion sources are easily interchangeable in most mass spectrometers. However, AP-MALDI suffers from less-than-optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal-to-noise (S/N) ratio gains observed when an on-chip dynamic preconcentration/focusing approach is coupled to AP-MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP-MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200–900 for hydrophobic substrates, and 150–400 for weak cation exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/μL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter-day reproducibility. The improved sensitivity allowed successful tandem MS sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP-MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads

NADP+-dependent farnesol dehydrogenase, a corpora allata enzyme involved in juvenile hormone synthesis

The synthesis of juvenile hormone (JH) is an attractive target for control of insect pests and vectors of disease, but the minute size of the corpora allata (CA), the glands that synthesize JH, has made it difficult to identify important biosynthetic enzymes by classical biochemical approaches. Here, we report identification and characterization of an insect farnesol dehydrogenase (AaSDR-1) that oxidizes farnesol into farnesal, a precursor of JH, in the CA. AaSDR-1 was isolated as an EST in a library of the corpora allata-corpora cardiaca of the mosquito Aedes aegypti. The 245-amino acid protein presents the typical short-chain dehydrogenase (SDR) Rossmann-fold motif for nucleotide binding. This feature, together with other conserved sequence motifs, place AaSDR-1 into the “classical” NADP+-dependent cP2 SDR subfamily. The gene is part of a group of highly conserved paralogs that cluster together in the mosquito genome; similar clusters of orthologs were found in other insect species. AaSDR-1 acts as a homodimer and efficiently oxidizes C10 to C15 isoprenoid and aliphatic alcohols, showing the highest affinity for the conversion of farnesol into farnesal. Farnesol dehydrogenase activity was not detected in the CA of newly emerged mosquitoes but significant activity was detected 24 h later. Real time PCR experiments revealed that AaSDR-1 mRNA levels were very low in the inactive CA of the newly emerged female, but increased >30-fold 24 h later during the peak of JH synthesis. These results suggest that oxidation of farnesol might be a rate-limiting step in JH III synthesis in adult mosquitoes

Parallel Spectral Acquisition with an Ion Cyclotron Resonance Cell Array

Mass measurement accuracy is a critical analytical figure-of-merit in most areas of mass spectrometry application. However, the time required for acquisition of high-resolution, high mass accuracy data limits many applications and is an aspect under continual pressure for development. Current efforts target implementation of higher electrostatic and magnetic fields because ion oscillatory frequencies increase linearly with field strength. As such, the time required for spectral acquisition of a given resolving power and mass accuracy decreases linearly with increasing fields. Mass spectrometer developments to include multiple high-resolution detectors that can be operated in parallel could further decrease the acquisition time by a factor of <i>n</i>, the number of detectors. Efforts described here resulted in development of an instrument with a set of Fourier transform ion cyclotron resonance (ICR) cells as detectors that constitute the first MS array capable of parallel high-resolution spectral acquisition. ICR cell array systems consisting of three or five cells were constructed with printed circuit boards and installed within a single superconducting magnet and vacuum system. Independent ion populations were injected and trapped within each cell in the array. Upon filling the array, all ions in all cells were simultaneously excited and ICR signals from each cell were independently amplified and recorded in parallel. Presented here are the initial results of successful parallel spectral acquisition, parallel mass spectrometry (MS) and MS/MS measurements, and parallel high-resolution acquisition with the MS array system

Biochemical, Molecular, and Functional Characterization of PISCF-Allatostatin, a Regulator of Juvenile Hormone Biosynthesis in the Mosquito Aedes aegypti

Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10(−9) M. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti