Cysteinyl peptide capture for shotgun proteomics: global assessment of chemoselective fractionation

Abstract

The complexity of cell and tissue proteomes presents one of the most significant technical challenges in proteomic biomarker discovery. Multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based shotgun proteomics can be coupled with selective enrichment of cysteinyl peptides (Cys-peptides) to reduce sample complexity and increase proteome coverage. Here we evaluated the impact of Cys-peptide enrichment on global proteomic inventories. We employed a new cleavable thiol-reactive biotinylating probe, N-(2-(2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenylethyl)phenoxy)acetamido)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (IBB), to capture Cys-peptides after digestion. Treatment of tryptic digests with the IBB reagent followed by streptavidin capture and mild alkaline hydrolysis releases a highly purified population of Cys-peptides with a residual S-carboxymethyl tag. Isoelectric focusing (IEF) followed by LC-MS/MS of Cys-peptides significantly expanded proteome coverage in Saccharomyces cerevisiae (yeast) and in human colon carcinoma RKO cells. IBB-based fractionation enhanced detection of Cys-proteins in direct proportion to their cysteine content. The degree of enrichment typically was 2-8-fold but ranged up to almost 20-fold for a few proteins. Published copy number annotation for the yeast proteome enabled benchmarking of MS/MS spectral count data to yeast protein abundance and revealed selective enrichment of cysteine-rich, lower abundance proteins. Spectral count data further established this relationship in RKO cells. Enhanced detection of low abundance proteins was due to the chemoselectivity of Cys-peptide capture, rather than simplification of the peptide mixture through fractionation.

Scheme 1. Synthesis of IBB

See “Experimental Procedures” for detailed description. Reagents: (a) TBDMSCl, Et₃N, THF, 86%; (b) 2-phenyl-1,3-dithiane, n-butyllithium, THF, 91%; (c) methyl bromoacetate, TBAF, THF, 93%; (d) 2,2′-(Ethylenedioxy)bisethylamine, MeOH, 75%; (e) biotin, CDI, DMF, 56%; (f) HgClO₄, CH₃CN/H2O, 69%; (g) iodoacetic acid, DCC, DMAP, 81%.

Scheme 2. (A) Chemical Structure of Base-Cleavable Cysteine-Reactive Probe (IBB), (B) Modification of Model Peptide (Ac-TpepC) by IBB and Hydrolysis of the Conjugate, and (C) Experimental Design for Evaluation of IBB Labeling and Streptavidin Capture of Cys Peptides

Consists of three functional elements: a thiol-reactive iodoacetamido group, a mild base-cleavable benzoin linker and biotin affinity tag. FT denotes non-Cys-peptides not retained on streptavidin. E denotes S-carboxymethyl Cys-peptides released by hydrolysis of IBB. G denotes complete mixture (“global fraction”) generated by hydrolysis of IBB-treated lysate before application to streptavidin. IBB labeling of cysteinyl thiols is done at pH 7, whereas streptavidin capture is done at pH 4.5. Elution/hydrolysis of IBB Cys-peptide conjugates is done at pH 8.

Figure 1

Reverse phase LC−MS total ion chromatograms for reaction products of Ac-TpepC and IBB under different conditions. The labeled peaks are I, Ac-TpepC; II, Ac-TpepC-IBB conjugate; III, IBB hydrolysis product; IV, IBB; V, S-carboxymethyl-Ac-TpepC. (A) Ac-TpepC; (B) Ac-TpepC-IBB conjugate formed by reaction of Ac-TpepC with IBB in 100 mM sodium phosphate, pH 7.0, containing 33.3% TFE at room temperature in dark for 20 min; (C) Ac-TpepC-IBB conjugate in 50 mM sodium phosphate buffer containing 3.67% TFE, pH 5.5 at 37 °C for 4 h; (D) Ac-TpepC-IBB conjugate in 50 mM sodium phosphate buffer containing 3.67% TFE, pH 6.5 at 37 °C for 4 h; (E) Ac-TpepC-IBB conjugate in 50 mM sodium phosphate buffer containing 3.67% TFE, pH 7.5 at 37 °C for 4 h; (F) Ac-TpepC-IBB conjugate pH 8.0, 50 mM ammonium bicarbonate at room temperature for 5 h; (G) Ac-TpepC-IBB conjugate in pH 4.5, 50 mM acetate buffer at room temperature in the dark overnight.

Figure 2

Relationship between IBB enrichment effect and Cys-peptide abundance for detected proteins. Proteins are ordered on the x-axis (“protein index”) in order of increasing fractional Cys-peptide content (Cys-peptide fraction), which is shown on the right y-axis of each plot. The red curves in (A) and (C) represent the theoretical Cys-peptide fraction (predicted Cys-peptides/predicted tryptic peptides) for each protein, whereas those in (B) and (D) represent the detected Cys-peptide fraction (detected Cys-peptides/detected tryptic peptides) for each protein. The black data points represent log₂-transformed ratios of spectral counts for detected proteins in the eluted (E) fraction to the global (G) fraction and are plotted as log₂ (E/G) (left y-axis). The listed R values are Spearman correlation coefficients for proteins with at least one predicted or detected Cys-peptide.

Figure 3

Venn diagram representation of proteins uniquely identified in yeast and RKO cells by Cys-peptide capture. Proteins identified in global (G) and eluted (E) fractions are shown. Note that 287 and 569 proteins were identified only in the E fractions and thus indicate the benefit of Cys-peptide capture.

Figure 4

Relationship between fractionation effect and Cys-peptide abundance in (A) yeast and (B) RKO cell proteins. Layout of plots is as for Figure 2, except that IEF subfractions 4, 5, and 6 (A) or IEF subfractions 4, 5, 6, and 7 (B) were substituted for the E fraction in IBB fractionated samples.

Figure 5

Impact of chemoselective fractionation with IBB on detection of yeast proteins annotated with TAP tag copy numbers. Proteins are grouped by copy number into four bins on the x-axis. One-way ANOVA analyses indicate significant differences in IBB enrichment effect based on protein spectral counts in global (G) fraction. Data points represent log₂ (E/G) for detected protein spectral counts in the eluted (E) fraction to the global (G) fraction. Means for each group are indicated by a long black line, and standard error of the mean (SEM) are indicated by short lines. *** Indicates p < 0.001.

Figure 6

Venn diagrammatic representation of enhanced identification of low abundance, Cys-containing proteins in yeast. The number of identifications from the Cys-peptide-containing eluted (E) fraction is inversely proportional to protein copy number.

Figure 7

Impact of chemoselective fractionation with IBB on detection of yeast proteins grouped by spectral counts. Proteins are grouped by spectral counts into five bins on the x-axis. One-way ANOVA analyses indicate significant differences in IBB enrichment effect based on protein spectral counts in global (G) fraction. Data points represent log₂ (E/G) for detected protein spectral counts in the eluted (E) fraction to the global (G) fraction. Means for each group are indicated by a long black line, and standard error of the mean (SEM) are indicated by short lines. *** Indicates p < 0.001.

Figure 8

Impact of chemoselective fractionation with IBB on detection of RKO proteins grouped by spectral counts. Proteins are grouped by spectral counts into five bins on the x-axis. One-way ANOVA analyses indicate significant differences in IBB enrichment effect based on protein spectral counts in global (G) fraction. Data points represent log₂ (E/G) for detected protein spectral counts in the eluted (E) fraction to the global (G) fraction. Means for each group are indicated by a long black line, and standard error of the mean (SEM) are indicated by short lines. *** Indicates p < 0.001.