Thermal proteome profiling of childhood ALL cell lines for proteoform detection
The complexity of the functional proteome extends significantly beyond the protein coding genome resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases, but challenging to perform systematically. Here, we apply thermal proteome profiling with deep peptide coverage to detect functional proteoforms in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detect 15,846 proteoforms, capturing differently spliced, post-translationally modified, and cleaved proteins expressed from 9,290 genes. We identify differential coaggregation of proteoform pairs and establish links to disease biology and drug sensitivity specific biomarkers. This approach provides a powerful and unique tool for systematic detection and functional annotation of proteoform groups.
Steps to reproduce
Cell lines were resuspended to a density of 100*10e6 cells/mL and distributed as 100 μL aliquots into 0.2-mL PCR tubes with a total of 8 aliquots per cell line. Each of the aliquots were heated for 3 min to the respective temperatures (41, 44, 47, 50, 53, 56, 59, 63°C), followed by a 3-min incubation time at RT. Afterwards, cells were flash-frozen in liquid nitrogen. Cells were thawed at 25°C and lysed by this freeze-thawing cycle repeated for another two times. Cell debris and precipitated proteins were removed by centrifugation at 21000 g and 4°C for 40 min. Supernatants were transferred to new tubes and protein concentrations were determined. Equal volumes of each condition that correspond to 100 μg protein in the 37°C sample were transferred to new tubes and subjected to the following digestion. First, the samples were diluted to contain 50 mM TEAB, 0.1% SDS and 5 mM TCEP. Reduction was performed at 65°C for 30 min. The samples were then cooled down to RT and alkylated with 15 mM of chloroacetamide for 30 min. The proteins were digested overnight with 1 to 40 Lys-C to protein-ratio and consecutively with Trypsin at a 1 to 25 enzyme to protein ratio. The digested peptides were labeled by TMTpro 16-plex using 0.6 mg of the respective label for each sample. Of note, the labelling efficiency was determined by LC-MS/MS before pooling of the samples. Two cell lines per TMTpro 16-plex set was used. After pooling, a sample clean-up step using a solid phase extraction (SPE strata-X-C, Phenomenex, Torrance, CA, USA) was performed and purified samples were dried in a vacuum centrifuge. Aliquots of approximately 400 µg peptides were pre-fractionated by means of HiRIEF using gel strips with pI range of 3-10 as well as 3,7-4,9 resulting in 42 and 72 fractions respectively. Collected fractions were dried using a vacuum centrifuge and subjected to an LC-MS/MS measurement.