Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale

Abstract

Described here is a chemo-selective enrichment strategy, termed the semitryptic peptide enrichment strategy for proteolysis procedures (STEPP), to isolate the semitryptic peptides generated in mass spectrometry-based proteome-wide applications of limited proteolysis methods. The strategy involves reacting the ε-amino groups of lysine side chains and any N-termini created in the limited proteolysis reaction with isobaric mass tags. A subsequent digestion of the sample with trypsin and the chemo-selective reaction of the newly exposed N-termini of the tryptic peptides with N-hydroxysuccinimide (NHS)-activated agarose resin removes the tryptic peptides from solution, leaving only the semitryptic peptides with one nontryptic cleavage site generated in the limited proteolysis reaction for subsequent LC-MS/MS analysis. As part of this work, the STEPP technique is interfaced with two different proteolysis methods, including the pulse proteolysis (PP) and limited proteolysis (LiP) methods. The STEPP-PP workflow is evaluated in two proof-of-principle experiments involving the proteins in a yeast cell lysate and two well-studied drugs, cyclosporin A and geldanamycin. The STEPP-LiP workflow is evaluated in a proof-of-principle experiment involving the proteins in two cell culture models of human breast cancer, MCF-7 and MCF-10A cell lines. The STEPP protocol increased the number of semitryptic peptides detected in the LiP and PP experiments by 5- to 10-fold. The STEPP protocol not only increases the proteomic coverage, but also increases the amount of structural information that can be gleaned from limited proteolysis experiments. Moreover, the protocol also enables the quantitative determination of ligand binding affinities.

Figure 1.

Schematic representation of the STEPP protocol workflow developed in this work.

Figure 2.

Schematic representation of the STEPP-PP workflow used in this work.

Figure 3.

Schematic representation of the STEPP-LiP workflow used in this work.

Figure 4.

Representative STEPP-PP data obtained in the CsA binding study. (A) Data from the 11 semi-tryptic peptides assayed from the known CsA binding protein, CPR1 (peptidyl-prolyl cis-trans isomerase). (B) Data from the 2 semi-tryptic peptides assayed from the known CsA binding protein, CPR3 (peptidyl-prolyl cis-trans isomerase C, mitochondrial). (C) Data from the 10 semi-tryptic peptides assayed from a non-hit protein, RPL38 (60S ribosomal protein L38). The dashed lines indicate the log₂(fold-change) values that are +/− 3σ deviations from the average log₂(fold-change) values of all the semi-tryptic peptides assayed in the CsA binding study.

Figure 5.

STEPP-PP data obtained in the geldanamycin binding experiment. (A) Data obtained on the 28 semi-tryptic peptides assayed HSP82 peptides in the geldanamycin binding experiment. The 4 hit peptides are shown in blue, red, yellow and green, other non-hit peptides are shown in grey. The dashed lines indicate the log₂(fold-change) values that are +/− 3σ deviations from the average log₂(fold-change) values of all the semi-tryptic peptides assayed in the geldanamycin binding study. (B) Schematic representation of the HSP82 geldanamycin binding domain with the geldanamycin bound (PDB ID: 1A4H). The four hit peptides from HSP82 (highlighted with the same colors as in A, respectively) all mapped to this known geldanamycin binding domain. (C) Data used to calculate the K_d value associated with the HSP82-geldanamycin complex. The data points represent the median data from the four hit peptides in (A). The solid line is the best fit of the data to equation S-1 (see Supplemental Text).

Figure 6.

Comparison of the STEPP-LiP and LiP data generated on HSP90AA1 (heat shock protein HSP 90-alpha) in this work and in reference 6 (respectively). (A) The amino acid positions and p-values of the 37 semi-tryptic peptides from HSP90AA1 isoform 1 that were assayed in the STEPP-LiP study of MCF-7 vs MCF-10A protein conformation are plotted. (B) The amino acid positions and p-values of the 4 semi-tryptic peptides from HSP90AA1 isoform 1 that were assayed in the LiP study of MCF-7 vs MCF-10A protein conformation in reference 6 are plotted. In both (A) and (B), the dashed lines correspond to a p-value = 0.05. The color of each data point indicates the MCF-7/MCF-10A TMT reporter ion intensity fold-change of the given semi-tryptic peptide, with more proteolytic susceptibility implicated by a greener color. (C) Schematic representations of the three-dimensional structures associated with the open and closed conformations of the HSP90 homodimer (generated from PDB ID: 2IOQ and 2CG9).^–