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The handle http://hdl.handle.net/1887/38868 holds various files of this Leiden University dissertation
Author: Heemskerk, A.A.M.
Title: Exploring the proteome by CE-ESI-MS Issue Date: 2016-04-28
Summary
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This thesis covers the progress that was made in the application of a porous sheathless interface in CE-ESI-MS analysis of protein digests. It has previously been shown that the porous sheathless interface could produce electrospray and separations at ultra- low flowrates and therefore had the potential to greatly improve the performance of CE-ESI-MS. Electrospray ionization at ultra-low flow rates has also shown to have significant advantages over conventional nano flow rates (±300 nl/min) not only regarding sensitivity but also minimizing ionization efficiency bias for specific compounds. The porous sheathless interface has shown to consistently generate a stable electrospray at flow rates below 10 nl/min and could therefore potentially produce the same ionization effects that were previously observed with regards to reduced ion suppression. Chapter 1 investigates the potential of the porous sheathless interface at ultra-low flow rates in reduction of ion suppression in the ionization of phosphorylated peptides. For these experiments five synthetic peptides with a constant amino acid sequence and varying numbers of phosphorylations (0-4) were obtained. The observed effect was a significant increase in sensitivity for the multiply phosphorylated peptides with the greatest improvement (factor 4) for the peptide containing 4 phosphorylations. The ionization efficiency of the interface was also shown in the analysis of IgG 1 derived glycopeptides.
Using a neutrally coated capillary the separation could be performed at flow rates below 10 nl/min and therefore large volume injection through transient isotachophoresis combined with ultra-low flow ionization resulted in a 20-fold improvement in sensitivity compared to the currently used platform (Chapter 2).
The separation at ultra-low flow rates is not only beneficial for the ionization process but equally important is the significant improvement in separation power that can be achieved using this approach. Chapter 3 shows the potential of a period of Zero-Flow separation combined with large volume injection before eventual mass spectrometry. It was found that 100% increase in peak capacity could be achieved using this approach with minimal deterioration of peak shape therefore maintaining sensitivity. Such improvements in peak capacity can directly translate in quite large improvements in numbers of identified peptides in bottom-up proteomics of complex sample.
Although CE-MS does not belong to the mainstream methods used in bottom-up proteomics, a number of groups have explored CE as an alternative or complementary technique to the current standard nano-LC-MS. To obtain a perspective of the history and applications in the field of CE-MS proteomics and more specifically CE-ESI-MS bottom-up proteomics two in-depth literature reviews were performed. The first review (Chapter 4) looked at the field of CE-MS bottom-up proteomics as a whole and the
developments made in recent years. This review covered both CE-MALDI and CE-ESI-MS including the varying interfaces that are available for this technique. As the review in Chapter 4 showed the promise of CE-ESI-MS for bottom-up proteomics a critical look was taken at all the required conditions for such a separation and analysis setup which resulted in the complete review presented in Chapter 5. In this review each aspect of the separation system is discussed separately and placed in the larger scope of developed applications to provide a novel user of CE-ESI-MS a frame of reference to start from. It was found that currently the best method of improving sensitivity is the use of large volume injections through a stacking approach which was therefore used throughout all experiments performed in this thesis.
Chapter 6 describes the development of a data processing workflow for the comparison of samples that are fractionated by SDS-PAGE. Because SDS-PAGE separates on basis of protein size/mass it is completely orthogonal to RPLC and CE separations making the fractions suitable to be analyzed by both techniques. A comparison of the two data sets would then show to what extent the two techniques are complementary in bottom-up up proteomics of more complex samples. The developed workflow showed excellent complementarity between the two strategies on both the identified peptide numbers and their hydrophobicity and peptide size. Moreover the data set from CE-MS identified more peptides and proteins.
The experiments in Chapter 7 show the application of the sheathless CE-ESI-MS platform to the analysis of human glomeruli. A strategy similar to the one used in Chapter 6 was applied to obtain in-depth proteomics knowledge on human glomeruli. Compared to the only previous in-depth experiment on human glomeruli, significantly more proteins were identified by our CE-MS platform. The analysis of laser micro dissected glomeruli from protocol needle biopsies is the ultimate goal in proteomics analysis of glomeruli.
An investigation was performed by using isolated glomeruli from cadaver kidneys as a model sample to determine the number of proteins that can be identified from a certain amount of material. It was found that material equivalent to only one glomerulus was enough to identify more than 100 proteins using the developed strategy.
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This thesis shows the promise of CE-ESI-MS using the porous sheathless interface for the analysis of post-translational modifications on proteins and for the analysis of protein digests. It was found that the low sample requirement and high sensitivity of CE-ESI-MS results in excellent sensitivity. Although, the sample loadability of the system is still a limitation, this is an engineering issue that should be solvable in the future. There are, therefore, strong indications that CE-ESI-MS with the porous sheathless interface can become a mainstream tool in the analytical environment.
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