Improving the Center Section Sequence Coverage of Large Proteins Using Stepped-Fragment Ion Protection Ultraviolet Photodissociation

J. Am. Soc. Mass Spectrom.

et al. 2023

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.

Section: ■ Results and Discussionmentioning

confidence: 89%

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

J. Am. Soc. Mass Spectrom.

et al. 2023

“…However, advanced modulation of the ion packet may be needed as benefits can be limited. 25 Ultimately, the scope and upper bounds of MALDI-UVPD is not currently known, and to date our UHMR Q Exactive HF has not detected a proteoform larger than 17 kDa from tissue. Further development of the instrument, and MALDI-UVPD will offer both increased sequence coverage and reduced experimental time, yet in the most basic form broad utility for MALDI applications has been demonstrated.…”

Section: Resultsmentioning

confidence: 90%

193 nm ultraviolet photodissociation for the characterization of singly charged proteoforms generated by MALDI

et al. 2022

Preprint

MALDI imaging allows for the near-cellular profiling of proteoforms directly from microbial colonies, or plant and mammalian tissues. Despite detecting hundreds of proteoforms, identification of unknowns with only intact mass information remains a distinct challenge, even at with high resolution 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 an UHMR HF Orbitrap. This platform permitted the high-resolution accurate mass measurement of not just terminal fragments, but also large internal fragments. The outlined workflow demonstrated the feasibility of high resolution top-down analysis of MALDI generated protein ions and the potential towards more comprehensive characterization of proteoforms in MALDI imaging applications.

“…However, advanced modulation of the ion packet may be needed as benefits can be limited. 26 Ultimately, the scope and upper bounds of MALDI-UVPD is not currently known, and to date our UHMR Q Exactive HF has not detected a proteoform larger than 17 kDa from tissue. Yet these small proteoforms can have important and diverse biological functions (i.e., signaling, metabolism, etc.…”

Section: Methodsmentioning

confidence: 90%

193 nm ultraviolet photodissociation for the characterization of singly charged proteoforms generated by MALDI

et al. 2023

Preprint

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 re-solved 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 an UHMR HF Orbitrap. This platform permitted the high-resolution 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 towards more comprehensive characterization of proteoforms in MALDI imaging applications.