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Evaluation and application of novel mass spectrometry-based proteomic workflows

Skoric, Sofija (2025) MOBN03 20242
Degree Projects in Molecular Biology
Popular Abstract
Why studying proteins is important

All our cells contain hereditary information, DNA, that allows the building blocks of our body, the cells, to know how to behave. However, the DNA is only the instruction book while proteins are the key players doing all the important work. We have to cope with many situations throughout our lives, such as stress, hunger and sickness among various others, and it is proteins that allow our bodies to respond adequately to these. Proteins can look very differently and have an immense number of important roles such as fixing our DNA when it’s damaged therefore keeping us healthy. However, sometimes such damages can go unnoticed and expand to multiple cells, possibly generating tumors. Understanding the... (More)
Why studying proteins is important

All our cells contain hereditary information, DNA, that allows the building blocks of our body, the cells, to know how to behave. However, the DNA is only the instruction book while proteins are the key players doing all the important work. We have to cope with many situations throughout our lives, such as stress, hunger and sickness among various others, and it is proteins that allow our bodies to respond adequately to these. Proteins can look very differently and have an immense number of important roles such as fixing our DNA when it’s damaged therefore keeping us healthy. However, sometimes such damages can go unnoticed and expand to multiple cells, possibly generating tumors. Understanding the mechanisms by which tumors arise and how they can be treated is crucial, therefore studying proteins is required. Investigating the full protein content of a cell, tissue or organism is called proteomics where cells are ruptured and proteins are broken down to its constituents, peptides, which are analyzed. Specifically, enzymes are used to break the peptide bonds within proteins to generate peptides, which can then be analyzed using ultra-sensitive and efficient instruments such as mass spectrometers.

In this master thesis, four different projects were conducted with the purpose to enhance various steps in the proteomic workflow. In project I, comparison of two different enzymes was done to see if a reduction of enzyme generated more peptide identifications. One of the enzymes outperformed the other and a reduction by half showed optimal results.

In many proteomic experiments mammalian cells are used, however, a common contaminant of these is the Mycoplasma bacteria which has the ability to alter the biological function of cells generating inaccurate experimental results. For this reason, project II utilized the high sensitivity of the mass spectrometer to detect mycoplasma peptides which were filtered to build a library of mycoplasma peptides. This library was used to search acquired data against when conducting experiments enabling early on mycoplasma detection if contamination has occurred.

Project III optimized the OneTip protocol, which allows proteomic studies of single cells. Here different enzyme concentrations, digestion times and cell suspension volumes compared to the standard protocol were tested to see which combination would acquire best results for low cell inputs. Overall, a reduction in enzyme concentration allowed for more peptide identifications while changing the remaining parameters did not show significant differences.

As our cells have the DNA encapsulated in the nucleus to separate it from the remaining cell compartment called the cytosol, techniques to separate the nuclear proteins from the cytosolic proteins exist. However, they are not applicable for many samples as it would be too time consuming and tedious. In project IV, a development of this method was done which can be used for many samples at the same time allowing studies of large number of samples utilizing separation of nuclear and cytosolic proteins. Overall, the proteomic workflow was enhanced to reduce its cost and material resources while allowing large scale proteomic studies.

Master’s Degree Project in Molecular Biology 60 credits 2025
Department of Biology, Lund University
Advisor: Ignacio Arribas Diez
Acrivon AB (Less)
Please use this url to cite or link to this publication:
author
Skoric, Sofija
supervisor
organization
course
MOBN03 20242
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9214808
date added to LUP
2025-11-03 13:58:59
date last changed
2025-11-03 13:58:59
@misc{9214808,
  author       = {{Skoric, Sofija}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Evaluation and application of novel mass spectrometry-based proteomic workflows}},
  year         = {{2025}},
}