![]() ![]() In BUP, exogenous proteases (e.g., trypsin) are used to cleave intact proteins into short peptides that are easier to detect with current LCMS instrumentation. Due to the large size of proteins, bottom-up proteomics (BUP) is the dominant approach for proteome characterization. ![]() Mass spectrometry (MS)-based proteomics is a powerful technique to measure proteins in biological samples, especially for mapping and quantifying post-translational modifications (PTMs).( Angel et al., 2012 Schaffer et al., 2019) The high resolving power of modern mass spectrometers, combined with various separation techniques (primarily liquid chromatography, LC) enables deep characterization of the proteome from complex samples. On-going technology developments are expected to further improve TDP coverage for more comprehensive high-throughput analysis of proteoforms. Taken together, our study demonstrates how untargeted TDP approach can provide unique insights into plant proteoform biology. Many of the proteins detected in both LCMS and MALDI-MSI are currently uncharacterized in UniProt: the PTM and spatial information presented here will be valuable in understanding their biological functions. A subset of peaks from the MALDI-MSI were assigned to proteoforms detected in TDP LCMS data based on matching accurate masses. Several detected proteoforms exhibited unique spatial distributions inside the infection zone and cortex, suggesting functional compartmentalization in these regions. In parallel, we collected high resolution MS imaging (MSI) data of intact proteins and biopolymers (<20 kDa due to current technical limitations) from sections of the soybean root nodules using matrix-assisted laser desorption/ionization (MALDI) coupled to high resolution Orbitrap. This included less common PTMs such as myristoylation, palmitoylation, cyanylation, and sulfation. Proteoforms with various PTMs and combinations thereof were identified using an unrestricted open modification search. Interestingly, these truncated proteoforms were considerably more abundant in the wildtype relative to the nifH- mutant, implicating protease activity as an important factor in nitrogen fixation. Leghemoglobin, the most abundant protein in the sample, existed in many truncated proteoforms. TDP captured 1648 proteoforms derived from 313 bacterial genes and 178 soybean genes. We performed TDP on soybean root nodules infected by the symbiotic Bradyrhizobium japonicum in both the wildtype bacterium and a nifH- mutant, which lacks the ability to fix nitrogen in the soybean root nodule. In contrast, top-down proteomics (TDP) directly characterizes intact proteins including all possible post-translational modifications (PTMs), thus offering unique insights into proteoform biology where combinations of individual PTMs may play important roles. Most well-established methods (known as bottom-up proteomics, BUP) employ an enzymatic digestion step to cleave intact proteins into smaller peptides for liquid chromatography (LC) mass spectrometry (MS) detection. ![]() Proteomic methods have been widely used to study proteins in complex biological samples to understand biological molecular mechanisms.
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