Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.
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We thank O. Schubert (UCLA) for insightful discussions. We also thank T. Lehmann for building the device to hold the sample tubes in the water bath and G. de Franceschi for contributing to the setup of the original LiP-MS protocol. P.P. is supported by an EU FP7-ERC Starting Grant (FP7-ERC-StG-337965), a 'Foerderungsprofessur' grant from the Swiss National Science Foundation (grant PP00P3_133670), and Promedica Stiftung (grant 2-70669-11). Y.F. is supported by an ETH Research Grant (grant no. 4412-1); I.P. is supported by an EMBO long-term fellowship (EMBO ALTF2014); and A.K. acknowledges SystemsX.ch for funding.
The authors declare no competing financial interests.
Integrated supplementary information
(A-D) Protein extracts from S. cerevisiae cells were subjected to the LiP-MS protocol with PK. LiP was conducted for one minute at a E/S of 1/100. After the LiP step, sodium deoxycholate (DOC) or urea were added to denature protein fragments (see conditions in Box 1) and complete digestion was obtained by the sequential addition of Lys-C and trypsin. The resulting peptides from LiP-treated and control samples were analyzed by LC-MS/MS. (A) The total number of proteins and (B) peptides, (C) the percent of missed-cleavage (MC) and (D) of half-tryptic (HT) peptides out of the total number of peptides identified are reported for the different treatments.
(A-D) Protein extracts from S. cerevisiae cells were subjected to the LiP-MS protocol with PK. LiP was conducted at an E/S of 1/100, using different incubation times as indicated. After the LiP step, sodium deoxycholate (DOC) was added to denature protein fragments as indicated in the protocol and complete digestion was obtained by the sequential addition of Lys-C and trypsin. The resulting peptides from LiP-treated and control samples were analyzed by LC-MS/MS. (A) The total number of proteins and (B) peptides, (C) the percent of missed-cleavage (MC) and (D) of half-tryptic (HT) peptides out of the total number of peptides identified are reported for the different treatments.
Crucial to the proper quenching of PK is obtaining consistent and maximal heat throughout the water bath. We recommend using a metallic water bath for optimal heat transfer. To hold sample tubes inside the water bath, we recommend using a homebuilt device such as the one shown in (A). Alternatively, floating racks such as the ones shown in B) can be used. Depending on their material, floating racks such as those shown in (B) may deteriorate with heat. We recommend measuring the temperature of the boiling water bath and ensuring that it is above 95°C to achieve complete inactivation of PK after the first proteolysis step. Water levels should be sufficient to submerge sample tubes above the sample level, but floating of tubes should be avoided. Note that the water level in the water bath will decrease over prolonged use with serial quenching of multiple samples or sample batches.
(A) Three-dimensional structure of horse myoglobin (Protein Data Bank entry 2frf) shown in a grey cartoon representation, which highlights its mainly alpha-helical secondary structure. Protein regions for which LiP peptides were identified after LiP of a sample containing apomyoglobin are displayed in yellow, while half-tryptic peptide ends are coloured in red. The bound heme molecule is represented as a multicolored ball and stick model. Note the two half-tryptic peptide ends embedded in the helix F next to the heme molecule. The two half-tryptic peptides are increasing in abundance or become detectable in samples containing apomyoglobin, relative to samples containing holomyoglobin, since in apomyoglobin the F helix is replaced by a locally unfolded segment, easily cleaved by PK (see also Feng et al., 2014). (B) LiP-MS analysis of core carbon metabolism upon a shift from glycolytic to gluconeogenic growth, adapted from Feng et al., 2014. A schematic representation of core carbon metabolism in S. cerevisiae, comprising the glycolytic pathway, the TCA and glyoxylate cycles, and the ethanol production branch is shown. Proteins are colored according to the results of LiP-MS and protein abundance measurements by LC-MS. Abundance changes are differences between levels in gluconeogenic relative to glycolytic growth. Only significant abundance changes are reported. Protein abundance changes were determined based on measurements conducted on the trypsin control sample. Changes in the LiP pattern were calculated from data obtained on the LiP-treated sample and after normalization for protein abundance changes. Reproduced with permission from Feng et al., Global analysis of protein structural changes in complex proteomes. Nat. Biotechnol. 32, 1036–1044 (2014), Nature Publishing Group.
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Schopper, S., Kahraman, A., Leuenberger, P. et al. Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry. Nat Protoc 12, 2391–2410 (2017). https://doi.org/10.1038/nprot.2017.100
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