The common concept in this thesis was to adapt and develop quantitative mass spectrometric assays focusing on reagents originating from the Human Protein Atlas project to quantify proteins in human cell lines, tissues and blood. The work is based around stable isotope labeled protein fragment standards that each represent a small part of a human protein-coding gene. This thesis shows how they can be used in various formats to describe the protein landscape and be used to standardize mass spectrometry experiments. The first part of the thesis describes the use of antibodies in combination with heavy stable isotope labeled antigens to establish a semi-automated protocol for protein quantification of complex samples with fast analysis time  (Paper~I). Paper II introduces a semi-automated cloning protocol that can be used to selectively clone variants of recombinant proteins, and highlights the automation process that is necessary for large-scale proteomics endeavors. This paper also describes the technology that was used to clone all protein standards that are used in all of the included papers.                       The second part of the thesis includes papers that focus on the generation and application of antibody-free targeted mass spectrometry methods. Here, absolute protein copy numbers were determined across human cell lines and tissues (Paper III) and the protein data was correlated against transcriptomics data. Proteins were quantified to validate antibodies in a novel method that evaluates antibodies based on differential protein expression across multiple cell lines (Paper IV). Finally, a large-scale study was performed to generate targeted proteomics assays (Paper V) based on protein fragments. Here, assay coordinates were mapped for more than 10,000 human protein-coding genes and a subset of peptides was thereafter used to determine absolute protein levels of 49 proteins in human serum.                       In conclusion, this thesis describes the development of methods for protein quantification by targeted mass spectrometry and the use of recombinant protein fragment standards as the common denominator. / <p>QC 20161013</p>

The designer proline-rich antimicrobial peptide (PrAMP) Chex1-Arg20 amide (ARV-1502) is active against Gram-negative and Gram-positive pathogens in differentmurine infection models when administered parenterally and possesses a wide therapeutic index. Here we studied the pharmacokinetics of ARV-1502 for the first time when administered intramuscularly or intravenously (IV) in Sprague Dawley rats and Beagle dogs. First, a specific and robust quantitation method relying on parallel reaction monitoring (PRM) using a high-resolution hybrid quadrupole-Orbitrap mass spectrometer coupled on-line to reversed-phase uHPLC was established and validated. The limit of detection was 2 ng/mL and the limit of quantitation was 4 ng/mL when spiked to pooled rat and dog plasma. When ARV-1502 was administered IV at doses of 75 and 250 μg/kg in dogs and rats, the plasma concentrations were 0.7 and 3.4μg/mL 2min post-administration, respectively. ARV-1502 plasma concentrations declined exponentially reaching levels between 2 and 4 ng/mL after 2 h. Intramuscular administration of 0.75 mg/kg in dogs and 2.5 mg/kg in rats resulted in a different pharmacokinetics profile. The plasma concentrations peaked at 15min post-injection at 1μg/mL (dogs) and 12μg/mL (rats) and decreased exponentially within 3 h to 4 and 16 ng/mL, respectively. The initial plasma concentrations of ARV-1502 and the decay timing afterwards indicated that the peptide circulated in the blood stream for several hours, at some point above the minimal inhibitory concentration against multidrug-resistant Enterobacteriaceae, with blood concentrations sufficient to suppress bacterial growth and to modulate the immune system.