New method to identify and explore functional proteoforms and their associations with drug response in childhood acute lymphoblastic leukemia

The paper examines melting behavior of proteins to define cases where portions of the protein melt differently. In these cases, the method can identify that the protein is likely to exist in multiple physical forms, called proteoforms. Therefore, a new perspective on variations between proteins can be interpreted. The method is applied in the context of childhood acute lymphoblastic leukemia cell lines, and is used to identify specific proteoforms associated with disease biology and drug response. This disease was selected as a proof of principle due to the need for improved precision therapies for patients.

New way of identifying protein variants

Proteins are responsible for execution of cellular processes and define what type of functions a cell is capable of performing. Beyond the wide extent of variation in genetic information, there are also many physical sources of protein variation, which include drug or biomolecule binding, relocalization, and other important modifications. Proteoforms that adopt variance beyond their genetic composition may have traits that open the door to wider evolution of adaptive protein functions, and could even be important reasons diseased cells are resistant or vulnerable to drug treatment. Understanding proteoforms and their functions is therefore crucial to understanding cell biology and disease processes, but it's a challenging task to detect proteoform differences technically and to know what differences to look for. This study introduces an untargeted way to identify many different new varieties of proteoforms simultaneously, for the first time including proteoform types that previously could only be studied in slower and more specialized ways.

The researchers used a panel of 20 childhood ALL cell lines and acquired mass-spectrometry based thermal stability measurements. They also applied a method to sort the protein fragments and improve their identification, which allowed unprecedented peptide coverage per protein. They used a graph-based analytical approach to analyze the data which allowed them to sort proteins into their co-existing proteoforms. They performed analyses to profile the type of biological change that contributed to important proteoforms. They also investigated their association of these proteoforms with the drug response of 378 cancer and investigational drugs where they could show that different proteoforms of a given protein correlated differently with drug response.

Important results for drug development and precision medicine

- In this study, we have developed a new method to identify separate physical versions of proteins that previously couldn't be differentiated from each other, and we did this based on their thermal stability says Rozbeh Jafari, senior researcher at the Department of Oncology-Pathology and last author of the paper.

- Being able to identify these differences improves interpretation of disease biology and drug response, and therefore provides a powerful and unique tool that sheds new light on the role of proteins and their proteoforms in ALL and other cancers. It has important implications for drug development and precision medicine, and could pave the way for more effective treatments. We will now continue to explore additional aspects and benefits of this method in more clinically relevant models”

The research was financed by Swedish Childhood Cancer Foundation, Swedish Research Council, Cancer Society Stockholm and the King Gustaf V Jubilee Fund, Dr. Åke Olsson Foundation for Hematological Research, Magnus Bergvalls Stiftelse and Felix Mindus Contribution to Leukemia research.

Publication

Deep thermal profiling for detection of functional proteoform groups
Nils Kurzawa, Isabelle Rose Leo, Matthias Stahl, Elena Kunold, Isabelle Becher, Anastasia Audrey, Georgios Mermelekas, Wolfgang Huber, André Mateus, Mikhail M. Savitski & Rozbeh Jafari
Nature Chemical Biology 20 March 2023