A New and Sensitive LC-MS Approach for PTM Characterization and High Sequence Coverage
Shiaw-Lin Wu; Jeongkwon Kim; William S. Hancock; Barry L. Karger;
Northeastern University, Boston, MA 02115
(ASMS 2005 Poster # 477)

Introduction
The most common mass spectrometric approaches for the characterization of proteins are based on either the analysis of their tryptic peptides (bottom-up) or the analysis of the intact proteins directly (top-down). In either strategy, the characterization of post-translational modifications, such as glycosylation and phosphorylation, is difficult due to either the size or the complexity of the analyte if analyzing intact proteins or the low detection sensitivity for tryptic peptides with glycosylation or phosphorylation in a mixture. We introduce a new approach, ERPA (extended range proteomic analysis), which combines the advantages of a reduction in the size and the complexity of samples, improved chromatographic and mass ionization efficiency, and the unique characteristics of a hybrid FTMS-linear ion trap.

Methods
The preferred approach includes (i) the use of enzymes which cut at less frequent sites than trypsin, i.e. Lys-C, to reduce the size and complexity of the analyte from an intact protein to fragments below 10,000 Daltons, (ii) high performance LC separation of the Lys-C fragments, and (iii) coupling of FTICR and a linear ion trap in a hybrid mass spectrometer. The FTICR cell provides a survey scan with high resolution (> 100,000) and accurate mass (> 2 ppm) to precisely determine the high charge-state precursors of the large peptides. Simultaneously, the linear ion trap provides fragmentation with a fast scan speed and high ion capacity for MS2 and MS3 fragmentation with collisionally-induced dissociation.

Preliminary Results
We have developed a new platform, extended range proteomic analysis (ERPA), for high sequence coverage of proteins with post-translational modifications. This ERPA platform combines advantages of the top-down and bottom-up proteomic approaches by analyzing peptides from approximately 500 to 10,000 Da. The small or large peptides are analyzed directly, with high charge states being resolved using Fourier transform ion cyclotron resonance (FTICR), followed by fragmentation in parallel in a linear ion trap mass spectrometer. This new LC-MS approach has been shown to characterize heavily modified phosphorylated and glycosylated proteins with significantly improved sensitivities (e.g. 10x to 100x) for phosphorylated or glycosylated peptides, relative to tryptic digest. The ERPA approach in the analysis of two proteins with different extents of phosphorylation and glycosylation: phosphorylated beta-casein (23 KDa), and glycosylated and phosphorylated epidermal growth factor receptor (180 KDa), shows nearly complete sequence coverage. In addition, the heavily modified phosphopeptides and glycopeptides in the mixtures are extensively characterized with high sensitivity (low to moderate fmol range). This approach should be directly applicable to the analysis of protein mixtures purified by immunoprecipitation or from gel spots, enabling extensive characterization of proteins of interest such as therapeutics or drug targets, using only a single automated on-line LC-MS system.