Differential exoprotease activities confer tumor-specific serum peptidome patterns

Abstract

Recent studies have established distinctive serum polypeptide patterns through mass spectrometry (MS) that reportedly correlate with clinically relevant outcomes. Wider acceptance of these signatures as valid biomarkers for disease may follow sequence characterization of the components and elucidation of the mechanisms by which they are generated. Using a highly optimized peptide extraction and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS-based approach, we now show that a limited subset of serum peptides (a signature) provides accurate class discrimination between patients with 3 types of solid tumors and controls without cancer. Targeted sequence identification of 61 signature peptides revealed that they fall into several tight clusters and that most are generated by exopeptidase activities that confer cancer type-specific differences superimposed on the proteolytic events of the ex vivo coagulation and complement degradation pathways. This small but robust set of marker peptides then enabled highly accurate class prediction for an external validation set of prostate cancer samples. In sum, this study provides a direct link between peptide marker profiles of disease and differential protease activity, and the patterns we describe may have clinical utility as surrogate markers for detection and classification of cancer. Our findings also have important implications for future peptide biomarker discovery efforts.

Figures

Figure 1. Unsupervised hierarchical clustering and principal component analysis of MS-based serum peptide profiling data derived from 3 groups of cancer patients and healthy controls.

(A) Serum samples from healthy volunteers and patients with advanced prostate, bladder, and breast cancer were prepared following the standard protocol. The 4 groups were randomized before automated solid-phase peptide extraction and MALDI-TOF MS. Spectra were processed and aligned using the Qcealign script (see Supplemental Methods). A peak list containing normalized intensities of 651 m/z values for each of the 106 samples was generated. Numbers indicate the number of patients and controls analyzed in the respective groups. (B) Unsupervised, average-linkage hierarchical clustering using standard correlation as a distance metrics between each cancer group and the control in binary format. The entire peak list (651 × 106) was used. Columns represent samples; rows are m/z peaks (i.e., peptides). Dendrogram colors follow the color coding scheme of A. The heat map scale of normalized ion intensities is from 0 (green) to 200 (red) with the midpoint at 100 (yellow). (C) Hierarchical clustering of the 3 cancer groups plus controls (as in B). (D) Principal component analysis (PCA) of the 3 cancer groups plus controls. Color coding is as in A. The first 3 principal components, which account for most of the variance in the original data set, are shown.

Figure 2. Feature selection and comparative analysis of serum peptide profiling data derived from 3 groups of cancer patients and healthy controls.

(A) The peak list was subjected to a Mann-Whitney U test for each individual cancer versus the control. Only peaks with adjusted P values of less than 0.00001 were passed through a second filter (median peak intensity > 500 units); a peak was selected if it passed the threshold in 1 cancer or in the control. (B) Venn diagrams show the number of peptides that passed both feature selection steps. The numbers shown outside the diagrams indicate the total number of peptides of a specific cancer group that were either up (Higher intensity) or down (Lower intensity). (C) Heat maps compare the selected features of the 3 cancer groups with controls in multiclass and binary formats. Columns represent samples (per group); rows are m/z peaks (not in numerical order). Peptides used in each binary comparison are the sum of those specifically higher and lower in each cancer group; the multiclass heat map contains the combined, nonredundant number of peptides. The multiclass, bladder, and breast heat map scales of normalized intensities range from 0 (green) to 500 (red) with the midpoint at 250 (yellow); those of the prostate map are from 0 (green) to 2,000 (red), with the midpoint at 1,000 (yellow).

Figure 3. MALDI-TOF mass spectral overlays of selected peaks derived from serum peptide profiling of 3 groups of cancer patients and healthy controls.

Spectra were obtained, aligned, and normalized as described in Methods and were displayed using the mass spectra viewer. Peptide ions have been selected to illustrate group-specific differences in normalized intensities, except for 2021.05, which is provided here as an example of the vast majority of peptide ions with intensities that were not statistically different between any 2 groups. The 24 overlays (not to scale) each show a binary comparison for all spectra from either the bladder cancer (n = 20; green), prostate cancer (n = 32; blue), or breast cancer patient group (n = 21; red) versus the control group (n = 33; yellow). They are arrayed so that an identical mass range window is shown for each of the 3 binary comparisons in which spectral intensities have been normalized and scaled to the same size. The monoisotopic mass (m/z) is shown for each peptide ion peak.

Figure 4. MALDI-TOF/TOF MS/MS identification of serum peptide 2305.

0 as a fragment of complement C4a. Peptides from a serum sample of a breast cancer patient were extracted and analyzed by MS and the ion of choice selected for MS/MS analysis, as described in Supplemental Methods. The fragment ion spectrum shown here was taken for a Mascot MS/MS ion search of the human segment of the NR database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein) and retrieved a sequence, GLEEELQFSLGSKINVKVGGNS ([MH]+ = 2305.19; Δ = 4 ppm), with a Mascot score of 38. b and y fragment ion series are indicated together with the limited sequences (arrows at top). Note that y ions originate at the C terminus and that the sequence therefore reads backwards (see direction of the arrows).

Figure 5. Serum peptide signatures for advanced prostate, bladder, and breast cancer.

Selected peptides identified by MALDI-TOF/TOF MS/MS are listed in clusters (ladders) of overlapping sequences, including 46 of the initial signature group of 68 (Figure 2 and Supplemental Table 2). m/z values are monoisotopic. Twenty-three additional peptides were positively matched to the existing clusters by hypothesis-driven, targeted MS/MS analysis. Overall, 61 entries had clear marker potential (adjusted P < 0.0002; Figure 6) for at least 1 cancer type and are color-coded blue (prostate cancer), green (bladder cancer), or red (breast cancer). Resulting signatures for the 3 cancers consist of 26 (prostate), 50 (bladder), and 25 (breast) peptide ions. Color-coded peptides have either higher (no filled circles) or lower (filled circles) differential ion intensities in a particular cohort of cancer samples compared with controls. C3f (m/z = 2021.05) and 1 member of the fibrinogen α cluster (m/z = 2553.01) gave comparable ion signals in all patient groups and control sera (see Figure 3, 2021, and Figure 6) and therefore represent effective internal standards (yellow). Six peptides (pink) were randomly observed. Residues in brackets were not experimentally observed but are shown to either indicate putative full-length sequences of the founder peptides and/or the positions of trypsin-like cleavage sites (Arg/Lys–Xaa).

Figure 6. Serum peptide signatures for advanced prostate, bladder, and breast cancer.

This table contains the same 69 entries as in Figure 5 plus additional details on the identified peptides (listed as m/z values), MS ion intensities, and signatures. The significance levels of 3 different Mann-Whitney U tests (columns 6–8) and of a multiclass Kruskal-Wallis test (column 9) are given. The actual signatures (blue, green, or red) are composed of entries that showed clear peptide ion marker potential (adjusted P < 0.0002) for at least 1 type of cancer. Adjusted P value is the overriding criterion, leading to final signatures of 26 (prostate), 50 (bladder), and 25 (breast) peptide ions (identical to those shown in Figure 5). The second column lists median intensities of each m/z peak in the control samples. Peak intensity ratios (columns 3–5) were calculated by dividing the median values of each m/z peak in each cancer group by the median value of the corresponding peak in the control samples. Ratios (r) for those peptides that are part of 1 or more signatures are shaded dark grey when the median signal is of higher intensity in a particular cancer (r ≥ 1.4) and lighter gray when it is lower (r ≤ 0.75). Norm., normalized.

Figure 7. Median ion intensities of serum peptides of selected sequence clusters relative to the corresponding values in the control group.

Median intensity for each peptide in each of the 3 cancer groups is plotted as the ratio versus the median intensity of the counterpart in the control group (r = patient/control). Ratios are plotted on a log scale ranging from 0.1 to 10. Bars pointing to the left (r < 1) or right (r > 1) indicate, respectively, lower or higher median intensities in a cancer group as compared with the control group. Peptides that didn’t show much difference in median ion intensity between patient and control groups map closely to or onto the center line (r = 1).

Figure 8. Study overview.

The diagram shows the approach used for development and validation of the 68-peptide ion signature and the prostate cancer signature consisting of 26 serum peptides with known sequence (blue in Figure 5). Numbers that are circled indicate total number of selected peptides at that stage of the study.

Figure 9. Independent set of prostate cancer serum samples for validation of established peptide signature biomarkers.

(A) Study design. See Figure 8 and Results. (B) Hierarchical cluster analysis of all spectra from PR1, PR2, and control groups. Either the 68 peptide ions with statistically significant intensity differences for the 3 binary comparisons (Figure 2) or 26 of the sequenced peptides that constitute the prostate cancer signature (blue in Figure 5) were used; the rest of the approximately 650 peptide ions were ignored. The heat map scale of normalized ion intensities ranges from 0 (green) to 2,000 (red) with the midpoint at 1,000 (yellow). (C) Principal component analysis of the PR1 and PR2 groups plus controls, based on the same peptide ions as in B. The first 3 principal components, accounting for most of the variance in the original data set, are shown.

Figure 10. Plasma exoproteases degrade synthetic C3f in a manner similar to proteolysis of the endogenous peptide (derived from C3 precursor) in serum.

A MALDI-TOF MS read-out of fresh plasma (top panel) indicates very low levels of small peptides except for bradykinin and desArg-bradykinin. After addition of synthetic C3f (1 pmol/μl plasma), an aliquot was immediately (i.e., after ~15–20 seconds) withdrawn, and another was withdrawn after 15 minutes. The sample was kept at room temperature at all times. The middle panel indicates removal of the C terminal Arg by a carboxypeptidase in a matter of seconds. C3f is then further degraded by the activity of aminopeptidases to result in a type of sequence ladder as endogenously present in serum. Brad (–R), bradykinin minus C-terminal Arg; R, Arg; RI, Arg-Ile; H, His; T, Thr; I, Ile; K, Lys; S, Ser.

Figure 11. Activity of serum proteases.

Many serum peptides are generated by a 2-step proteolytic process. When used in the proper combinations, 1 or more selected members of 6–12 different clusters create diagnostic signatures in the form of ion intensities measured by direct MALDI-TOF MS that can predict cancer and cancer type. Amino acids are color coded to represent sequence clusters of C3f (left) or FPA (right), which are just 2 examples of all the observed clusters.