Extended Range Proteomic Analysis (ERPA):  A New and Sensitive LC−MS Platform for High Sequence Coverage of Complex Proteins with Extensive Post-translational ModificationsComprehensive Analysis of Beta-Casein and Epidermal Growth Factor Receptor (EGFR)

Abstract

We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (> 100 000) and accurate mass (<2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate >95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.

Glycoproteomics Analysis of Human Liver Tissue by Combination of Multiple Enzyme Digestion and Hydrazide Chemistry

“…An alternative method is the digestion of glycoproteins by Lys-C to generate a small number of peptides than tryptic fragments and the resultant larger peptides is analyzed by high resolution Fourier transform ion cyclotron resolution mass spectrometer. 18 However, it is not easy to identify large peptides if low resolution ion trap mass spectrometer is used. Multiple enzyme digestion strategy has been applied to the analysis of other modifications.…”

Development of a Novel Polyclonal Antibody-Based Immunoassay for the Quantitation of Non-Albumin Urinary Proteins

“…Proteins eluted from the gel slices and aliquots of the albumin-Trf-IgG-absorbed urinary protein preparation were digested with trypsin and endoproteinase Lys-C (Lys-C) for 1 hour at 37 o C, and lyophilized. Digestions of proteins eluted from gel slices [24] and from aliquots of the albumin-Trf-IgG-absorbed urinary protein preparation [25] were performed as previously described. LC-MS/MS analysis of digested urinary protein and gel slice preparations of urinary proteins was performed by Transitions Therapeutics, Inc. (Toronto, Ontario, Canada) and the Keck Biotechnology Resource Laboratory at Yale University (New Haven, CT, USA), respectively.…”

What does the future hold for top down mass spectrometry?

“…The concept of Middle Down mass spectrometry has been in place for a while now [14], but it has not been until recent work by the Karger research group that this methodology was recognized as having potential as a new type of high-throughput proteomics platform. Wu et al showed that this Middle Down MS strategy termed Extended Range Proteomic Analysis could be used not only to obtain better sequence coverage than Bottom Up MS [81], but also could be performed under the same chromatographic conditions, and at similar sensitivities as Bottom Up MS. This group has gone on to improve large peptide separations and Middle Down MS analysis for the characterization of low levels of peptides carrying diverse kinds of PTMs [82].…”