Gel-Assisted Proteome Position Integral Shift Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Caspase 3 Substrates

Here, we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics and demonstrates its utility in studying proteolytic events in programmed cell death. With gel-assisted proteome position integral shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: (i) a negative MW shift; (ii) an elevated ratio in a pair of a semitryptic and tryptic peptide, (iii) a negative shift in the standard deviation of MW estimated for different peptides, and (iv) a negative shift in skewness of the same data. Of these proteins, 58 molecules were previously unreported caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, the GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.

Low-number passages (<10) were used for the experiments.
Staurosporine Treatment and Cell Lysis.HeLa cells were cultured in 75 cm 2 flasks for 20 h, subsequently treated with 300 nM staurosporine (STS) or vehicle (DMSO) for 4 h in four biological replicates.Cells were washed twice with phosphate buffered saline (PBS) and lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher) supplemented with 1% protease inhibitors (Roche).The cellular lysates were centrifuged at 12,000 rpm for 10 min at 4°C and the soluble fraction was collected.The protein concentration in the lysate was measured using Pierce BCA kit (Thermo Fisher).

SDS-PAGE and Gel Excision.
Cell lysate was diluted using M-PER Mammalian Protein Extraction Reagent for all samples to the same protein concentration.The electrophoresis was performed on NuPAGE 4-12% Bis-Tris Mini Protein Gel (Thermo Fisher) with two wells in MES Running Buffer under reduced conditions at 150 V for 60 min using the XCell SureLock system (Thermo Fisher). 100 µg of protein were loaded onto the gel for each sample.
One STS-treated sample and one DMSO-treated sample were processed in the same tank.Novex Sharp Pre-Stained Protein Standard (Thermo Fisher) was used as a ladder.After electrophoresis the gels were washed and excised diagonally into two parts (A and B), after which each part of the gel was cut into 1 × 1 mm cubes and transferred into 5 mL LoBind tubes.The gel cubes were centrifuged, followed by sequential washes with ammonium bicarbonate/acetonitrile (1:1, v/v) and acetonitrile, each for 10 min.During the acetonitrile incubation, the gel pieces exhibited shrinkage and opacity.Subsequently, all liquid was removed.
Reduction and Alkylation.25 mM of dithiothreitol (DTT) (Sigma-Aldrich) in 50 mM ammonium bicarbonate was added to fully immerse gel pieces.The samples were then incubated at 56 °C for 30 min.After cooling to room temperature (RT), any remaining liquid was removed.Following this, acetonitrile was added and incubated for 10 min at RT, after which all liquid was removed.Subsequently, 50 mM iodoacetamide (IAA) (Sigma-Aldrich) was added, and the samples were incubated at RT in darkness for 1 h.Then acetonitrile was used to shrink the gel pieces for 10 min with subsequent liquid removal.
Trypsin Digestion.Each sample was rehydrated at 4 °C for 30 min by adding the same volume of 10 ng/µL trypsin in 50 mM ammonium bicarbonate containing 0.01% ProteaseMAX Surfactant (Promega).During rehydration, the absorbance of the solution was checked, and additional trypsin solution was added to ensure that gel pieces immersed completely.Subsequent incubation was performed for 2 h at 37°C with agitation at 200 rpm.Condensate from the tube walls was collected by centrifugation at 12000 × g for 10 s.The digestion solution with extracted peptides was transferred into new 2 mL tubes, and trypsin was inactivated by adding trifluoroacetic acid (TFA) (Sigma-Aldrich) to a final concentration of 0.5%.
TMT-labeling.Samples were cleaned up using Sep-Pak cartridges (Waters) and dried in a DNA 120 SpeedVac Concentrator (Thermo Fisher).The dried peptide samples were reconstituted in 50 mM EPPS buffer (pH 8.5).
Acetonitrile (ACN) was added to achieve a final concentration of 30%.After cutting each gel lane into two pieces A and B, four replicates of DMSO-and STS-treated HeLa cells (eight samples) produced 16 sub-samples, one set of TMTpro 16plex was used for labeling these 16 sub-samples as shown in Figure 1g.TMTpro 16plex reagents (Thermo Fisher) were added at a 5:1 w/w ratio to each sample, followed by a 2 h-incubation at RT.The TMT labeling reaction was quenched by the addition of 0.5% hydroxylamine.All 16 TMT-labelled samples were combined, acidified with TFA, subjected to clean-up using Sep-Pak cartridges (Waters), and dried in a DNA 120 SpeedVac Concentrator (Thermo Fisher).
High-pH Fractionation.Peptide separation for deeper proteome analysis was carried out using an Ultimate 3000 HPLC system (Thermo Fisher) equipped with a Xbridge Peptide BEH C18 column (25 cm × 2.1 mm, particle size 3.5 μm, pore size 300 Å; Waters) operating at a flow rate of 200 μL/min.Fractionation was achieved through a binary solvent system comprising 20 mM NH4OH in H2O at pH 10 (solvent A) and 20 mM NH4OH in acetonitrile (solvent B).The elution profile was programmed as follows (data for % of solvent B): an initial gradient from 2% to 23% over 42 min, followed by a rapid increase to 52% within 4 min, further elevation to 63% in 2 min, and a subsequent isocratic hold at 63% for 5 min.The elution process was monitored by UV absorbance at 214 nm.A total of 96 fractions, each containing a 100 μL aliquot, were collected.Fractions were subsequently combined in a sequential order (e.g.1,25,49,73), providing a total of 24 composite fractions.
t-tests for the two treatment groups.The p-value adjustment was following the Benjamini and Hochberg method, employed for multiple hypothesis adjustment 1 : the peptides were ranked in descending order according to the number of peptides attributed to each protein, and p-values were subsequently adjusted by multiplying by their respective ranks.Given the follow-up validation, false discovery rate (FDR) of ≤10% was determined to be acceptable for proteins with significant MW shifts (FDR was defined as the number of proteins with significant MW increase divided by the total number of proteins with both significant MW increase and decrease).

Figure S1 .
Figure S1.Calibration curves converting B/A values to MW for the MS3 data set.(a) to (d) Data calibration curves for the four replicates of DMSO-treated HeLa cells, respectively.(e) to (h) Data calibration curves for the four replicates of STS-treated HeLa cells, respectively.

Figure S2 .
Figure S2.Calibration curves converting B/A values to MW for the MS2 data set.(a) to (d) Data calibration curves for the four replicates of DMSO-treated HeLa cells, respectively.(e) to (h) Data calibration curves for the four replicates of STS-treated HeLa cells, respectively.

Figure S3 .
Figure S3.Protein MW estimations from peptide B/A ratios for the MS2 data set.(a) Correlation of B/A ratios with MW for all 7433 proteins.(b) Calibration curves for all eight gels.(c) CV distribution of B/A-calculated protein MW values.(d) Error distribution of B/A-calculated protein MW values.

Figure S4 .
Figure S4.Volcano plot of proteins with significant MW shifts in the MS2 data set.With p values adjusted by peptides number-based multiple hypothesis correction, volcano plot shows 155 proteins significantly shift to lower MW (red) while 25 proteins significantly shift to higher MW (blue).

Figure S5 .
Figure S5.Standard deviation (SD) analysis for identifying caspase substrate candidates.(a) The empirical formula SD = 0.086MW 1.29 is fitted to the SD-MW plot for DMSO-treatment, with residual standard error of 13 kDa.(b) The empirical formula SD = 0.086MW 1.23 is fitted to the SD-MW plot with residual standard error 10 kDa for STS-treatment.(c) Volcano plot for 3384 proteins with ≥ 7 peptides from STS-treated HeLa cells with proteins showing significantly decreased (red) and increased (blue) SD.

Figure S6 .
Figure S6.Protein MW estimation from peptide B/A values for SD and skewness analysis.(a) A pseudogel with one protein's MW distribution across the four replicates in DMSO-and STS-treated HeLa cells.(b) A pseudogel with peptide-derived MW estimations of three proteins in STS-treated HeLa cells.

Figure S7 .
Figure S7.Skewness analysis for identifying caspase substrate candidates.Volcano plot for 1569 proteins with ≥ 13 peptides showing in STS-treated HeLa cells proteins with significantly decreased (red) and increased (blue) skewness.

Table S1 .
Validated caspase 3 candidate substrates.The proteins at No. 1 to 26 were found in literature.The 58 novel caspase 3 substrates were at No. 27 to 84.