Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR-Orbitrap Detection

Zemaitis, Kevin; Veličković, Dušan; Kew, Will; Fort, Kyle L.; Reinhardt-Szyba, Maria; Pamreddy, Annapurna; Ding, Yanli; Kaushik, Dharam; Sharma, Kumar; Makarov, Alexander; Zhou, Mowei; Paša‐Tolić, Ljiljana

doi:10.1021/acs.analchem.2c01034

Cited by 15 publications

(19 citation statements)

References 69 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Prior to analysis the tissue sections were desiccated under 61 kPa of vacuum for 30 min and then washed in fresh solutions of 70% ethanol for 30 s, 100% ethanol for 30 s, Carnoy's solution (6:3:1 v/v ethanol/chloroform/glacial acetic acid) for 2 min, 100% ethanol for 30 s, water with 0.2% TFA for 15 s, and immediately washed in 100% ethanol for 30 s. Samples were then dried by stream of nitrogen gas prior to direct tissue acidification to improve signal intensity. The protocol was described in detail elsewhere (Zemaitis et al, 2022), briefly, a HTX Technologies M5 Sprayer (Chapel Hill, NC, United States) was used to apply 5% (v/v) acetic acid in 50% ethanol directly prior to application of MALDI matrix (2,5dihydroxyacetophenone, DHA) at a coverage of 277 μg/cm 2 for protein desorption/ionization. After the matrix was applied it was recrystallized with 5% (v/v) aqueous acetic acid at 38.5 °C prior to analysis and dried for 3.5 min, using an apparatus similar to that previously reported.…”

Section: Maldi Proteoform Imagingmentioning

confidence: 99%

“…Detailed operation of this prototype instrument is described elsewhere, where ions were detected from tissue up to ~17 kDa with isotopic resolution. (Zemaitis et al, 2022) Briefly, the instrument was set to acquire over a m/z range of 3,250 to 20,000 at a preset resolution of 240 k, this resulted in an observed experimental resolution of 35 k at m/z 11,300 with 500 laser shots per pixel. Spectra were acquired with a raster of 15 µm to achieve cellular spatial resolution.…”

Section: Maldi Proteoform Imagingmentioning

confidence: 99%

“…In parallel, we interrogated spatial heterogeneity of proteoforms using matrix-assisted laser desorption/ionization (MALDI) MSI coupled to high resolution Orbitrap (Zemaitis et al, 2022) on thin tissue sections. The detected proteoforms were either concentrated in the infection zone, at the outer cortex, or nearly uniformly distributed across the tissue.…”

Section: Introductionmentioning

confidence: 99%

“…Currently, the standard TDP methods used here are limited to small proteins up to 20-30 kDa. (Huguet et al, 2019;Su et al, 2022;Zemaitis et al, 2022). We also discussed new developments in TDP instrumentation that will gradually bridge the gap and allow full integration with BUP data.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Discovery top-down proteomics in symbiotic soybean root nodules

Zhou

Fulcher²,

Zemaitis³

et al. 2022

Front. Anal. Sci.

Self Cite

View full text Add to dashboard Cite

Proteomic methods have been widely used to study proteins in complex biological samples to understand biological molecular mechanisms. 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. 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. 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. TDP captured 1648 proteoforms derived from 313 bacterial genes and 178 soybean genes. Leghemoglobin, the most abundant protein in the sample, existed in many truncated proteoforms. 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. Proteoforms with various PTMs and combinations thereof were identified using an unrestricted open modification search. This included less common PTMs such as myristoylation, palmitoylation, cyanylation, and sulfation. 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. Several detected proteoforms exhibited unique spatial distributions inside the infection zone and cortex, suggesting functional compartmentalization in these regions. A subset of peaks from the MALDI-MSI were assigned to proteoforms detected in TDP LCMS data based on matching accurate masses. 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. Taken together, our study demonstrates how untargeted TDP approach can provide unique insights into plant proteoform biology. On-going technology developments are expected to further improve TDP coverage for more comprehensive high-throughput analysis of proteoforms.

show abstract

Section: Maldi Proteoform Imagingmentioning

confidence: 99%

Section: Maldi Proteoform Imagingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Discovery top-down proteomics in symbiotic soybean root nodules

Zhou

Fulcher²,

Zemaitis³

et al. 2022

Front. Anal. Sci.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Despite the popularity of MALDI for protein analyses, application and outcomes of intact protein mass spectrometry imaging (MSI) are limited due to the challenge of detecting and annotating protein ions. While time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) are commonly used mass analyzers that have both been applied to analyze intact proteins, high-mass-resolving-power measurements by FTICR, and recently ultra-high-mass range (UHMR) Orbitrap, , offer advantages over TOF for more precise identification of proteoforms, distinct forms of proteins potentially harboring multiple post-translational modifications (PTMs). , …”

Section: Introductionmentioning

confidence: 99%

193 nm Ultraviolet Photodissociation for the Characterization of Singly Charged Proteoforms Generated by MALDI

Zemaitis

Zhou

Kew

et al. 2023

J. Am. Soc. Mass Spectrom.

Self Cite

View full text Add to dashboard Cite

MALDI imaging allows for the near-cellular profiling of proteoforms directly from microbial, plant, and mammalian samples. Despite detecting hundreds of proteoforms, identification of unknowns with only intact mass information remains a distinct challenge, even with high mass resolving power and mass accuracy. To this end, many supplementary methods have been used to create experimental databases for accurate mass matching, including bulk or spatially resolved bottom-up and/or top-down proteomics. Herein, we describe the application of 193 nm ultraviolet photodissociation (UVPD) for fragmentation of quadrupole isolated singly charged ubiquitin (m/z 8565) by MALDI-UVPD on a UHMR HF Orbitrap. This platform permitted the highresolution accurate mass measurement of not just terminal fragments but also large internal fragments. The outlined workflow demonstrates the feasibility of top-down analyses of isolated MALDI protein ions and the potential toward more comprehensive characterization of proteoforms in MALDI imaging applications.

show abstract