Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody. Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity. Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field

Protein microarrays for validation of affinity binders

Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents.QC 20111117Development and applications of protein microarraysThe Swedish Human Proteome Resource (HPR) progra

Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody. Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity. Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field

A Deep Learning Receiver for Non-Linear Transmitter

Non-linearity of wireless transceivers, specifically power amplifier (PA) non-linearity, could pose major limitations towards having high throughput, and cost and energy efficient wireless communication systems. Such limitations from the PA is typically compensated in the transmitter, e.g. by applying power back-off or performing digital-pre-distortion (DPD) aiming to linearize the transmitter. However, applying PA power back-off leads to lower energy efficiency, and lower output power, and hence lower coverage; and performing DPD results in higher complexity of the transmitters. This paper presents an alternative approach based on a receiver method to perform signal detection in the presence of distortions due to PA non-linearity. We propose a receiver technique using artificial neural networks (ANN) to compensate for the PA non-linearity at the receiver side. The paper presents link-level simulation results using pre-trained neural network models based on synthesized training data. The simulation results confirm that the designed receiver can tolerate higher distortions, hence allow the PA output power back-off to be reduced, leading to higher output power improving coverage, spectral efficiency, energy efficiency, and throughput.HexaX_WP

High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals. In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study

A systems study of the future waste management system in Borås : Part of the project: "Thermal and biological waste treatment in a systems perspective"

Finansierat av SP Sveriges Tekniska Forskningsinstitut Projektnummer WR-21Thermal and biological waste treatment in a systems perspectiv

A systems study of the waste management system in Gothenburg : Part of project: "Thermal and biological waste treatment in a systems perspective"

Finansiär SP Sveriges Tekniska Forskningsinstitut Projektnummer WR-21Termisk och biologisk avfallsbehandling i ett systemperspekti

Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry.

Today immunoassays are widely used in veterinary medicine, but lack of species specific assays often necessitates the use of assays developed for human applications. Mass spectrometry (MS) is an attractive alternative due to high specificity and versatility, allowing for species-independent analysis. Targeted MS-based quantification methods are valuable complements to large scale shotgun analysis. A method referred to as parallel reaction monitoring (PRM), implemented on Orbitrap MS, has lately been presented as an excellent alternative to more traditional selected reaction monitoring/multiple reaction monitoring (SRM/MRM) methods. The insulin-like growth factor (IGF)-system is not well described in the cat but there are indications of important differences between cats and humans. In feline medicine IGF-I is mainly analyzed for diagnosis of growth hormone disorders but also for research, while the other proteins in the IGF-system are not routinely analyzed within clinical practice. Here, a PRM method for quantification of IGF-I, IGF-II, IGF binding protein (BP) -3 and IGFBP-5 in feline serum is presented. Selective quantification was supported by the use of a newly launched internal standard named QPrEST™. Homology searches demonstrated the possibility to use this standard of human origin for quantification of the targeted feline proteins. Excellent quantitative sensitivity at the attomol/μL (pM) level and selectivity were obtained. As the presented approach is very generic we show that high resolution mass spectrometry in combination with PRM and QPrEST™ internal standards is a versatile tool for protein quantitation across multispecies