Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications

Malinovska, Liliana; Cappelletti, Valentina; Kohler, Devon; Piazza, Ilaria; Tsai, Tsung-Heng; Pepelnjak, Monika; Stalder, Patrick; Dörig, Christian; Sesterhenn, Fabian; Elsässer, Franziska; Kralickova, Lucie; Beaton, Nigel; Reiter, Lukas; Souza, Natalie de; Vitek, Olga; Picotti, Paola

doi:10.1038/s41596-022-00771-x

Cited by 41 publications

(49 citation statements)

References 30 publications

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“…These properties, equipped to alter the structure of the protein, can also influence the digestion efficiency of PK. In principle, flexible and accessible regions (such as loops or unstructured domains) of a protein are much more susceptible to time-limited digestion by PK, in comparison to folded or aggregated regions . Therefore, the alteration of the PK digestion efficiency can provide the context of structural flexibility and accessibility.…”

Section: Resultsmentioning

confidence: 99%

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer’s Disease

Lu,

Wang,

Liu

et al. 2023

Anal. Chem.

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High-throughput quantitative analysis of protein conformational changes has a profound impact on our understanding of the pathological mechanisms of Alzheimer’s disease (AD). To establish an effective workflow enabling quantitative analysis of changes in protein conformation within multiple samples simultaneously, here we report the combination of N,N-dimethyl leucine (DiLeu) isobaric tag labeling with limited proteolysis mass spectrometry (DiLeu-LiP-MS) for high-throughput structural protein quantitation in serum samples collected from AD patients and control donors. Twenty-three proteins were discovered to undergo structural changes, mapping to 35 unique conformotypic peptides with significant changes between the AD group and the control group. Seven out of 23 proteins, including CO3, CO9, C4BPA, APOA1, APOA4, C1R, and APOA, exhibited a potential correlation with AD. Moreover, we found that complement proteins (e.g., CO3, CO9, and C4BPA) related to AD exhibited elevated levels in the AD group compared to those in the control group. These results provide evidence that the established DiLeu-LiP-MS method can be used for high-throughput structural protein quantitation, which also showed great potential in achieving large-scale and in-depth quantitative analysis of protein conformational changes in other biological systems.

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Section: Resultsmentioning

confidence: 99%

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer’s Disease

Lu,

Wang,

Liu

et al. 2023

Anal. Chem.

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show abstract

“…O’Reilly and Rappsilber review cross-linking mass spectrometry (CLMS/XLMS), which uses bifunctional bioconjugation reagents to create covalent links between proximal amino acids . Limited proteolysis-mass spectrometry (LiP-MS) is a relatively robust and easy-to-use method for probing protein structure as well as small molecule-protein binding, using proteolytic enzymes to map the proteolytic vulnerabilities of peptide bonds. ,,− …”

Section: Advanced Topics and Further Readingmentioning

confidence: 99%

An Introduction to Mass Spectrometry-Based Proteomics

Shuken

2023

J. Proteome Res.

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Mass spectrometry is unmatched in its versatility for studying practically any aspect of the proteome. Because the foundations of mass spectrometry-based proteomics are complex and span multiple scientific fields, proteomics can be perceived as having a high barrier to entry. This tutorial is intended to be an accessible illustrated guide to the technical details of a relatively simple quantitative proteomic experiment. An attempt is made to explain the relevant concepts to those with limited knowledge of mass spectrometry and a basic understanding of proteins. An experimental overview is provided, from the beginning of sample preparation to the analysis of protein group quantities, with explanations of how the data are acquired, processed, and analyzed. A selection of advanced topics is briefly surveyed and works for further reading are cited. To conclude, a brief discussion of the future of proteomics is given, considering next-generation protein sequencing technologies that may complement mass spectrometry to create a fruitful future for proteomics.

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“…Limited proteolysis was performed as described previously. 26,48 In brief, samples were treated with proteinase K (PK) under controlled conditions (enzyme:substrate ratio of 1:100 for 5min, LiP samples) or with ddH2O (control samples). PK was thermally inactivated at 99 C for 5min.…”

Section: Western Blotmentioning

confidence: 99%

“…The generated peptides were desalted using C18 columns (The Nest Group Inc.) according to the manufacturer's protocol and analyzed as described previously. 48,49 For data dependent acquisition (DDA) replicates were pooled and LiP and control samples were injected independently. For data independent acquisition (DIA), each replicate a sample was injected.…”

Section: Western Blotmentioning

confidence: 99%

Calreticulin mutations affect its chaperone function and perturb the glycoproteome

et al. 2022

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Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications

Cited by 41 publications

References 30 publications

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer’s Disease

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer’s Disease

An Introduction to Mass Spectrometry-Based Proteomics

Calreticulin mutations affect its chaperone function and perturb the glycoproteome

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