Online Nanoflow RP−RP-MS Reveals Dynamics of Multicomponent Ku Complex in Response to DNA Damage

Abstract

Tandem affinity purification (TAP) coupled with mass spectrometry has become the technique of choice for characterization of multicomponent protein complexes. While current TAP protocols routinely provide high yield and specificity for proteins expressed under physiologically relevant conditions, analytical figures of merit required for efficient and in-depth LC-MS analysis remain unresolved. Here we implement a multidimensional chromatography platform, based on two stages of reversed-phase (RP) separation operated at high and low pH, respectively. We compare performance metrics for RP-RP and SCX-RP for the analysis of complex peptide mixtures derived from cell lysate, as well as protein complexes purified via TAP. Our data reveal that RP-RP fractionation outperforms SCX-RP primarily due to increased peak capacity in the first dimension separation. We integrate this system with miniaturized LC assemblies to achieve true online fractionation at low (e5 nL/min) effluent flow rates. Stable isotope labeling is used to monitor the dynamics of the multicomponent Ku protein complex in response to DNA damage induced by γ radiation.

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

“…In recent years, a different 2D-LC methodology has been developed, based on the RP separation under basic pH (pH 10) conditions in the first dimension and a low-pH RP separation in the second dimension. [26][27][28][29][30][31][32][33][34][35][36][37][38][39] In all reported work using capillary chromatography (e.g., 300 μm ID columns), [26][27][28][29][30][31][32][33][34][35][36] the first LC dimension was operated in an off-line mode using fraction collection, followed by excess organic solvent evaporation mAbs Fig. 2B) from the second dimension (lowpH) separations in four consecutive injections (experiments).…”

“…26,27,30,31 This observation prompted much interest in coupling two RP columns, operated at two pH extremes (pH 10 and pH 2.5), as a 2D chromatographic system for peptide separation, using online or off-line configurations. [26][27][28][29][30][31][32][33][34][35][36][37][38][39] Although the coupling of high-pH RP/low-pH reversed-phase separations was shown to be less orthogonal than the classical SCX/RP multidimensional system for the separation of complex peptide mixtures in proteomic experiments, 30 the separation resolution offered by the high-pH RP in the first chromatographic dimension is far superior to the SCX separation. RP separation elutes peptides almost equally over the entire retention window (trapezoidal distribution of peptides) allowing for a greater spread of peptides across the same number of fractions.…”

Online Nanoflow Multidimensional Fractionation for High Efficiency Phosphopeptide Analysis

“…These limitations have led to the development of numerous other strategies for fractionation of phosphopeptides including strong anion exchange chromatography (17,22), continuous pH gradients (29), ERLIC (30,31), and hydrophilic interaction chromatography (19), all utilizing RP chromatography for the second dimension separation. In this report we describe two approaches for fractionation of phosphopeptides that build upon our recent work (32) in coupling true, nanoflow chromatography with reversed phase-reversed phase (RP-RP) fractionation (28). First we utilize aliphaticfunctionalized quarternary amines as ion pairing agents to improve retention of phosphopeptides in the first dimension RP separation performed at high pH.…”

DNA Ends Alter the Molecular Composition and Localization of Ku Multicomponent Complexes

“…To date, Ku protein partners include the RNA helicase RHA and several heterogeneous ribonucleoproteins (13), the Werner Syndrome associated protein WRN (15)(16)(17), poly-(ADP-ribose) polymerase I PARP-1 (17,18), the yin-yang transcription factor YY1 (19), histone H2AX (20), and telomerase (12,14). Binary protein-protein interactions with Ku70 were also delineated by two-hybrid screen (21), whereas mass spectrometry-based approaches have been used to identify multicomponent protein complexes that copurify with Ku (22,23). These data provide a useful inventory of Ku protein partners, although the dynamics of specific interactions in response to exogenous or endogenous stimuli remain unknown.…”