Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions

Abstract

The study of isolated protein complexes has greatly benefited from recent advances in mass spectrometry instrumentation and quantitative, isotope labeling techniques. The comprehensive characterization of protein complex components and quantification of their relative abundance relies heavily upon maximizing protein and peptide sequence information obtained from MS and tandem MS studies. Recent work has shown that using a metalloendopeptidase, Lys-N, for proteomic analysis of biological protein mixtures produces complementary protein sequence information compared with trypsin digestion alone. Here, we have investigated the suitability of Lys-N proteolysis for use with MALDI mass spectrometry to characterize the yeast Arp2 complex and E. coli PAP I protein interactions. Although Lys-N digestion resulted in an average decrease in protein sequence coverage of approximately 30% compared with trypsin digestion, CID analysis of singly-charged Lys-N peptides yielded a more extensive b-ions series compared with complementary tryptic peptides. Taking advantage of this improved fragmentation pattern, we utilized differential (15)N/(14)N guanidination of Lys-N peptides and MALDI-MS/MS analysis to relatively quantify the changes in PAP I associations due to deletion of sprE, previously shown to regulate PAP I-dependent polyadenylation. Overall, this Lys-N/guanidination integrative approach is applicable for functional proteomic studies utilizing MALDI mass spectrometry analysis, as it provides an effective and economical mean for relative quantification of proteins in conjunction with increased sensitivity of detection and fragmentation efficiency.

Figure 1. Comparison of protein sequence coverage following trypsin and Lys-N digestion

(A) A representative SDS-PAGE gel from the immunopurification of Arp2-GFP from yeast. (B) The protein sequence coverages obtained following trypsin (top) and Lys-N (bottom) digestions are represented for the eight proteins from the isolated Arp2 complex. The total numbers of amino acids, R and K residues are indicated. The cleavage sites for trypsin and Lys-N are represented by vertical lines. Filled gray segments represent peptides with an m/z > 4000 that would not be detected by our MALDI LTQ Orbitrap instrument. Detected peptides are represented as black bars for unmodified peptides, and gray bars for peptides that are modified by oxidation of methionine. Projections of the observed peptides and corresponding sequence coverages are indicated. (C) Protein sequence coverage is plotted against the percentage of cleavage sites for the Arp2-isolated proteins following trypsin (open diamonds) and Lys-N (filled circles) digestions.

Figure 2. MALDI IT CID of trypsin- and Lys-N-derived peptides

Equivalent peptides from BSA, Arp2, and Arp3 following trypsin (left column) or Lys-N (right column) digestion were selected for MALDI CID MS/MS analysis. The m/z values for the selected peptides were (A) 1304.70, (B) 973.450, (C) 1603.72 and (D) 1337.66.

Figure 3. Guanidination improves sensitivity of detection and fragmentation of singly-charged ions

(A) The MALDI MS spectra of trypsin or Lys-N digested BSA are illustrated for the m/z 1100–1800 range. T_R⁺_n indicates arginine-containing tryptic peptides, while the T_K⁺_n indicates lysine-containing peptides. All the peptides derived from Lys-N digestion (denoted L⁺_n) contain lysine residues, and therefore are modified by guanidination. 50 fmol of tryptic β-casein peptides were spiked in the samples derived from the Lys-N digestion, serving as internal standards. The [M+H]⁺ peak at m/z 1383.70 from β-casein was included as a reference (labeled IS). (B) MALDI IT CID of T⁺₅₉, L⁺₄₁ BSA peptides (m/z 1346.71) and a selected Arp2 peptide (m/z 1645.72) are illustrated as representative MS/MS spectra. (C) MALDI IT CID of a Lys-N-derived Arp2 peptide (m/z 1383.75 unmodified and 1425.75 guanidinated) containing an N-terminus lysine and C-terminus arginine. K* represents homoarginine.

Figure 4. Integrating Lys-N digestion and guanidination for relative quantification studies

(A) Workflow of the relative quantification procedure. Proteins isolated with PAP I-GFP from E. coli cells were separated by 1D SDS PAGE, divided into equal aliquots and digested with trypsin or Lys-N. Peptides from the wild-type or ΔsprE cells were derivatized by guanidination with heavy (¹⁵N) or light (¹⁴N) labeled O-methylisourea, combined and analyzed using a MALDI LTQ Orbitrap. The intensities of the resulting doublets at MS or MS/MS levels of analysis were used for relative quantification. (B) Protein sequence coverage, number of lysine and arginine residues in PAP I and SrmB proteins. (C) Relative quantification of the association between PAP I-GFP and SrmB in ΔsprE cells. The ΔsprE peptides were normalized relative to the wild-type peptides. At least four peptides were used for each quantification. (D) Representative MALDI IT CID spectra of PAP I and SrmB peptides following trypsin and Lys-N digestions. Precursor ions were selected using an isolation window of ±6 mass units, which was centered at the midpoint m/z between the light and heavy monoisotopic peaks (m/z 1716.78 for illustrated PAP I peptide and m/z 933.47 and 1132.58 for the two SrmB peptides from trypsin and Lys-N digestions, respectively). The quantifications were performed utilizing the intensities of the monoisotopic fragment peaks averaging the resulting ratios from a minimum of 4 unique peptides. Inserts illustrate examples of y or b ion doublets used for relative quantification in trypsin or Lys-N studies, respectively.