Charge State Dependent Fragmentation in Top-Down Analysis by RP-LC-MS/MS Using a Standard Protein Mix

Introduction

Analytical labs that serve as a core facility are often tasked with performing LC-MS analysis of intact proteins from various internal and/or external research groups. The most widely used approaches for protein identification and characterization are enzymatic digestion followed by LC-MS/MS of digested peptides—i.e., bottom-up proteomics. A complementary approach to identify and characterize protein samples, which is referred to as top-down proteomics, involves tandem mass spectrometry of intact protein ions. Critical factors that affect structural information from MS/MS product ions of an intact protein include ion activation approach, ion polarity, modification of the ion, and protein ion charge state. In this study, we have examined charge state dependent fragmentation of several well-defined proteins present in a standard test mix.

Methods

RPLC-MS

Column: BIOshell A400 Protein C4, 1.0 mm x 15 cm, 3.4 µm; 60 °C

Mobile Phases: A: Water + 0.1% TFA; B: ACN + 0.1% TFA

Gradient: 20 - 60 %B; 0 - 20 min; Total run time 30 min, 90 µL/min

Handling: 0.5 M Ammonium bicarbonate / 0.5 M TCEP, 65 °C, 1 hr

LCMS System: Waters Acquity coupled to Thermo QE+ Orbitrap; Collision Energy 20-25 eV

Data collection and analysis were performed with Xcalibur, BioPharma Finder 3.0, and ProSight Lite

Protein Composition

Lysozyme: 17 basic amino acid residues (11 R + 6 K), 4 S-S bonds

Carbonic Anhydrase: 27 basic amino acid residues (9 R + 18 K)

Studied Parameters
  • S-S reduction
  • Collision energy
  • Protein charge state
  • Isolation window
  • BPF search strategies
Protein Charge State Categories

High: Number of Charges > Number of Basic Sites (R+K)

Intermediate: Number of Charges ≈ Number of Basic Sites (R+K)

Low: Number of Charges < Number of Basic Sites (R+K)

Summary

  • S-S reduction before MS/MS experiment showed more protein sequence coverage
  • Wide isolation window showed less sequence coverage and various unidentified peaks
  • The maximum amide bond cleavages was found for intermediate charge states ~(R+K) with prominent contributions from C-terminal Aspartic Acid and N-terminal Proline
  • Combining MS/MS peak lists from all charge states yielded the highest sequence coverage from the search

Non-reduced MSRT2 UV215nm chromatogram separating nine intact proteins

Figure 1. Non-reduced MSRT2 UV215nm chromatogram separating nine intact proteins using a BIOshell™ A400 Protein C4 column 1.0 mm × 15 cm, 3.4 µm at 90 µL/min. The insets show mass spectra of reduced Lysozyme and Carbonic Anhydrase (CA), which were used as model proteins for Top-Down optimization.

 

Effect of S-S Reduction

Effect of S-S Reduction - non-reducedEffect of S-S Reduction - reduced

Effect of Collision Energy

Effect of Collision Energy 30eVEffect of Collision Energy 20eV

Effect of Isolation Window

Effect of Isolation Window - 550 DaEffect of Isolation Window - 2 Da

Figure 2. MS/MS spectra of Lysozyme demonstrating optimization of sample handling, collision energy, and isolation window to maximize sequence coverage for top-down analysis. Green colored parameters were considered optimized for the model proteins evaluated.

 

Lysozyme
R + K = 17

Carbonic Anhydrase
R + K = 27

a) MS/MS Spectra

Deconvoluted MS/MS Spectra - LysozymeDeconvoluted MS/MS Spectra - Carbonic Anhydrase

Parent Ion: [M+17H]17+
Parent Ion: [M+27H]27+

b) Deconvoluted MS/MS Spectra

Deconvoluted MS/MS Spectra - LysozymeDeconvoluted MS/MS Spectra - Carbonic Anhydrase

c) Fragment Maps

Lysozyme Fragment MapsCarbonic Anhydrase Fragment Maps

36% Sequence Coverage
22% Sequence Coverage

d) Percent Sequence Coverage

Lysozyme Percent Sequence CoverageCarbonic Anhydrase Percent Sequence Coverage

Figure 3. Results from model proteins, lysozyme and carbonic anhydrase: a) MS/MS spectra, b) deconvoluted spectra, c) fragment maps of selected charge state, and d) percent sequence coverage from different charge states.

Materials