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Proteins are the molecular building blocks of all living organisms. They are the functioning actors in metabolism and communication, and they give specific architecture and purpose to all tissues. Their abundance is dynamic and can be interpreted and associated to different physiological states or diseases. Blood plasma, the liquid component of blood, is a proximal fluid to all organs in the human body. As a consequence, it contains an immense amount of information about wide variety of biological processes. This makes it a great sample to study but also demanding due to the complexity and dynamic range of protein concentrations. A promising technology capable of extracting the information is targeted proteomics. Especially in combination with heavy labelled standards it combines liquid chromatography and tandem mass spectrometry (LC-MS/MS) into state-of-the-art quantification on an absolute scale. This toolbox is capable of detecting the smallest perturbations thanks to its precision and multiplexing capability. In this thesis, I describe our efforts to develop and use a novel platform for targeted proteomics suitable for clinical applications with high demands on accuracy and reliability. In Paper I, a deep dive into the resource of standards coming from The Human Protein Atlas was performed. It was possible to identify their great analytical performance and generate LC-MS/MS coordinates for over 25,000 proteotypic peptides from over 10,000 proteins. A proof-of-concept experiment was designed based on the screening results where 23 proteins in blood plasma were absolutely quantified. The data allowed us to identify distinct clusters of individuals based on their LPA, APOE, SERPINA5 and TFRC levels. Paper II directly builds on the findings from Paper I and applies them on a well-defined cohort of 94 individuals whose blood plasma samples were collected over one-year period in four different time-points. The longitudinal protein profiling allowed to assess the vari, Proteiner är de molekylära byggstenar som bygger upp alla levande organismer. De utgör både funktionella och strukturella komponenter i vävnader och deltar i aktiviteter som metabolism och kommunikation. Deras nivåer varierar och beror på kroppens fysiologiska tillstånd, men också på olika sjukdomar. Blodplasma, vilket är den flytande komponenten av blod, är den vätska som är i kontakt med alla människokroppens organ. Detta gör blodplasma till en utmärkt källa att studera då den innehåller information som representerar de olika biologiska processer som sker. Det är dock svårt att studera proteiner i blod på grund den höga komplexiteten som kommer från stora koncentrationsskillnader hos olika protein. Riktad proteomik är en teknologi som trots detta kan tillgodogöra sig denna typ av information. Tekniken möjliggör träffsäker koncentrationsbestämning genom att använda sig av tunga isotopinmärkta standarder som mäts med hjälp av vätskekromatografi i kombination med tandemmasspektrometri. Även mycket små skillnader i proteinkoncentrationer kan upptäckas med tekniken tack vare den höga kvantitativa träffsäkerheten. Analyserna kan utföras parallellt och flera analyter kan mätas samtidigt (multiplex). I detta examensarbete beskriver jag våra insatser för att utveckla och använda en helt ny teknologisk plattform inom området för riktad proteomik som vi har utvecklat för kliniska tillämpningar som ofta associeras med mycket höga krav på noggrannhet och pålitlighet. I Artikel I genomfördes en rigorös djupdykning för att analysera en resurs av proteinstandarder som producerats inom The Human Protein Atlas. Studien beskriver experimentella kromatografiska koordinater för över 25 000 proteotypiska peptider från 10 000 individuella proteinstandarder. För att påvisa användbarheten kvantifierades och koncentrationsbestämdes även 23 proteiner i blodplasma. Resultatet möjliggjorde att specifika kluster av personer kunde urskiljas baserat på deras LPA-, APOE-, SERPINA5- och TFRC-nivåe, QC 2022-05-16

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed., QC 20220530

Spike-in of standards of known concentrations used in proteomics-based workflows is an attractive approach for both accurate and precise multiplexed protein quantification. Here, a quantitative method based on targeted proteomics analysis of plasma proteins using isotope-labeled recombinant standards originating from the Human Protein Atlas project has been established. The standards were individually quantified prior to being employed in the final multiplex assay. The assays are mainly directed toward actively secreted proteins produced in the liver, but may also originate from other parts of the human body. This study included 21 proteins classified by the FDA as either drug targets or approved clinical protein biomarkers. We describe the use of this multiplex assay for profiling a well-defined human cohort with sample collection spanning over a one-year period. Samples were collected at four different time points, which allowed for a longitudinal analysis to assess the variable plasma proteome within individuals. Two assays toward APOA1 and APOB had available clinical data, and the two assays were benchmarked against each other. The clinical assay is based on antibodies and shows high correlation between the two orthogonal methods, suggesting that targeted proteomics with highly parallel, multiplex analysis is an attractive alternative to antibody-based protein assays., QC 20210203

The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (<= 60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINAS, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development., QC 20190730