Basicranial and otic region anatomy of protoceras celer and the phylogenetic position of the family protoceratidae (order: artiodactyla)

Bibliography: p. 64-68Some pages are in colour

Methods of Protein Sample Preparation Using Digital Microfluidics and Gas-Phase Techniques with Detection by Mass Spectrometry

Biological samples are complicated, having many different components at a wide variety of concentrations. Some means to simplify these samples while leaving the discrete components unaltered is required. Small structural differences can render a protein unusable role. Mass spectrometry is a valuable tool to characterize proteins but the number of components in these samples present a challenge for accurate detection. Sample preparation and separation methods are effective tools to alleviate this challenge In this dissertation, I present methods for simplifying protein samples for analyze with mass spectrometry using liquid and gas phase methods. In the first chapter, I review some sample preparation options. In Chapter 2, I present a microscale method for high abundance protein depletion using digital microfluidics and functionalized magnetic particles. This is a fast, effective, and multiplexed method for reducing the interference caused from proteins like serum albumin and immunoglobulins resulting in a 4-fold improvement in signal to noise for a low abundant protein. Chapter 3 extends this methodology by employing the same fluid and particle handling technique to immunoprecipitation. This chapter introduces the use of a pre-concentration method called pre-concentration using liquid intake by paper (P-CLIP), a promising ‘world-to-chip’ interface for many digital microfluidic applications. In Chapter 4, gas-phase vapours are used to simplify analysis of peptides by electrospray mass spectrometry. Acetonitrile vapour suppresses in-source fragmentation of fragile ions by clustering with fragile ions resulting in improved limits of detection. Chapter 5 presents a union of liquid chromatography and differential mobility spectrometry for characterization of biopharmaceutical proteins. This new combination, called DMS-SWATH, increases MS/MS sequence coverage for proteins and alleviates problems found in traditional analysis by mass spectrometry and differential mobility spectrometry. Overall this work presents important steps forward in miniaturizing and ameliorating the use of mass spectrometry for the characterization of proteins.Ph.D.2019-07-11 00:00:0

A digital microfluidic interface between solid-phase microextraction and liquid chromatography-mass spectrometry

We introduce a method to couple solid-phase microextraction (SPME) with HPLC-MS using digital microfluidics (DMF). In the new system, SPME fibers are used to extract analytes from complex sample solutions, after which the analytes are desorbed into solvent droplets in a DMF device. The open geometry of DMF allows straightforward insertion of SPME fibers without requiring a complicated interface, and automated droplet manipulation enables multiplexed processing of the fibers. In contrast to other multiplexed SPME elution interfaces, the low volumes inherent to DMF allow for pre-concentration of analytes prior to analysis. The new SPME-DMF-HPLC-MS method was applied to the quantification of pg/mL-level free steroid hormones in urine. We propose that this new method will be useful for a wide range of applications requiring cleanup and pre-concentration with convenient coupling to high-performance analytical techniques

Digital Microfluidic Platform for Human Plasma Protein Depletion

Many important biomarkers for disease diagnosis are present at low concentrations in human serum. These biomarkers are masked in proteomic analysis by highly abundant proteins such as human serum albumin (HSA) and immunoglobulins (IgGs) which account for up to 80% of the total protein content of serum. Traditional depletion methods using macro-scale LC-columns for highly abundant proteins involve slow separations which impart considerable dilution to the samples. Furthermore, most techniques lack the ability to process multiple samples simultaneously. We present a method of protein depletion using superparamagnetic beads coated in anti-HSA, Protein A, and Protein G, manipulated by digital microfluidics (DMF). The depletion process was capable of up to 95% protein depletion efficiency for IgG and HSA in 10 min for four samples simultaneously, which resulted in an approximately 4-fold increase in signal-to-noise ratio in MALDI-MS analysis for a low abundance protein, hemopexin. This rapid and automated method has the potential to greatly improve the process of biomarker identification

Digital Microfluidic Platform for Human Plasma Protein Depletion

Many important biomarkers for disease diagnosis are present at low concentrations in human serum. These biomarkers are masked in proteomic analysis by highly abundant proteins such as human serum albumin (HSA) and immunoglobulins (IgGs) which account for up to 80% of the total protein content of serum. Traditional depletion methods using macro-scale LC-columns for highly abundant proteins involve slow separations which impart considerable dilution to the samples. Furthermore, most techniques lack the ability to process multiple samples simultaneously. We present a method of protein depletion using superparamagnetic beads coated in anti-HSA, Protein A, and Protein G, manipulated by digital microfluidics (DMF). The depletion process was capable of up to 95% protein depletion efficiency for IgG and HSA in 10 min for four samples simultaneously, which resulted in an approximately 4-fold increase in signal-to-noise ratio in MALDI-MS analysis for a low abundance protein, hemopexin. This rapid and automated method has the potential to greatly improve the process of biomarker identification

Digital Microfluidics for Immunoprecipitation

Immunoprecipitation (IP) is a common method for isolating a targeted protein from a complex sample such as blood, serum, or cell lysate. In particular, IP is often used as the primary means of target purification for the analysis by mass spectrometry of novel biologically derived pharmaceuticals, with particular utility for the identification of molecules bound to a protein target. Unfortunately, IP is a labor-intensive technique, is difficult to perform in parallel, and has limited options for automation. Furthermore, the technique is typically limited to large sample volumes, making the application of IP cleanup to precious samples nearly impossible. In recognition of these challenges, we introduce a method for performing microscale IP using magnetic particles and digital microfluidics (DMF-IP). The new method allows for 80% recovery of model proteins from approximately microliter volumes of serum in a sample-to-answer run time of approximately 25 min. Uniquely, analytes are eluted from these small samples in a format compatible with direct analysis by mass spectrometry. To extend the technique to be useful for large samples, we also developed a macro-to-microscale interface called preconcentration using liquid intake by paper (P-CLIP). This technique allows for efficient analysis of samples >100× larger than are typically processed on microfluidic devices. As described herein, DMF-IP and P-CLIP-DMF-IP are rapid, automated, and multiplexed methods that have the potential to reduce the time and effort required for IP sample preparations with applications in the fields of pharmacy, biomarker discovery, and protein biology

Analysis on the Go: Quantitation of Drugs of Abuse in Dried Urine with Digital Microfluidics and Miniature Mass Spectrometry

We report the development of a method coupling microfluidics and a miniature mass spectrometer, applied to quantitation of drugs of abuse in urine. A custom digital microfluidic system was designed to deliver droplets of solvent to dried urine samples and then transport extracted analytes to an array of nanoelectrospray emitters for analysis. Tandem mass spectrometry (MS/MS) detection was performed using a fully autonomous 25 kg instrument. Using the new method, cocaine, benzoylecgonine, and codeine can be quantified from four samples in less than 15 min from (dried) sample to analysis. The figures of merit for the new method suggest that it is suitable for on-site screening; for example, the limit of quantitation (LOQ) for cocaine is 40 ng/mL, which is compatible with the performance criteria for laboratory analyses established by the United Nations Office on Drugs and Crime. More importantly, the LOQ of the new method is superior to the 300 ng/mL cutoff values used by the only other portable analysis systems we are aware of (relying on immunoassays). This work serves as a proof-of-concept for integration of microfluidics with miniature mass spectrometry. The system is attractive for the quantitation of drugs of abuse from urine and, more generally, may be useful for a wide range of applications that would benefit from portable, quantitative, on-site analysis

Direct Interface between Digital Microfluidics and High Performance Liquid Chromatography–Mass Spectrometry

We introduce an automated method to facilitate in-line coupling of digital microfluidics (DMF) with HPLC-MS, using a custom, 3D-printed manifold and a custom plugin to the popular open-source control system, DropBot. The method was designed to interface directly with commercial autosamplers (with no prior modification), suggesting that it will be widely accessible for end-users. The system was demonstrated to be compatible with samples dissolved in aqueous buffers and neat methanol and was validated by application to a common steroid-labeling derivatization reaction. We propose that the methods described here will be useful for a wide range of applications, combining the automated sample processing power of DMF with the resolving and analytical capacity of HPLC-MS

Direct Interface between Digital Microfluidics and High Performance Liquid Chromatography–Mass Spectrometry

We introduce an automated method to facilitate in-line coupling of digital microfluidics (DMF) with HPLC-MS, using a custom, 3D-printed manifold and a custom plugin to the popular open-source control system, DropBot. The method was designed to interface directly with commercial autosamplers (with no prior modification), suggesting that it will be widely accessible for end-users. The system was demonstrated to be compatible with samples dissolved in aqueous buffers and neat methanol and was validated by application to a common steroid-labeling derivatization reaction. We propose that the methods described here will be useful for a wide range of applications, combining the automated sample processing power of DMF with the resolving and analytical capacity of HPLC-MS

A digital microfluidic interface between solid-phase microextraction and liquid chromatography–mass spectrometry

We introduce a method to couple solid-phase microextraction (SPME) with HPLC-MS using digital microfluidics (DMF). In the new system, SPME fibers are used to extract analytes from complex sample solutions, after which the analytes are desorbed into solvent droplets in a DMF device. The open geometry of DMF allows straightforward insertion of SPME fibers without requiring a complicated interface, and automated droplet manipulation enables multiplexed processing of the fibers. In contrast to other multiplexed SPME elution interfaces, the low volumes inherent to DMF allow for pre-concentration of analytes prior to analysis. The new SPME-DMF-HPLC-MS method was applied to the quantification of pg/mL-level free steroid hormones in urine. We propose that this new method will be useful for a wide range of applications requiring cleanup and pre-concentration with convenient coupling to high-performance analytical techniques