Analysis of E.coli Tryptic Digest and Intact Protein Using the Agilent 1290 Infi nity 2D-LC Solution with Diode Array Detection and Q-TOF LC/MS

Significance of the topic

Proteomic analysis of complex biological samples requires exceptional separation power to resolve hundreds of proteins and thousands of resulting peptides. Comprehensive two dimensional LC provides greatly enhanced peak capacity and selectivity by combining orthogonal chromatographic modes.

Study objectives and overview

This work demonstrates the use of the Agilent 1290 Infinity 2D LC Solution integrating strong cation exchange and reversed phase separations in the first and second dimensions. Diode array detection and an Agilent 6530 Accurate Mass Q TOF LC MS detector were applied to both E coli tryptic digest peptides and intact protein mixtures under identical hardware and column configurations. The goal is to maximize peak capacity and achieve confident identification of proteolytic peptides and intact proteins.

Methodology and instrumentation

• Sample preparation Urea denaturation reductive alkylation and tryptic digestion of E coli lysate
• First dimension SCX chromatography at pH 3 or pH 5 10 mM phosphate gradient plus NaCl elution on 2.1 x 250 mm SCX column at 65 microliters per minute
• Second dimension RPLC on a 4.6 x 50 mm C18 column at 3 to 3.5 milliliters per minute using 0.1 percent phosphoric or formic acid in water and acetonitrile gradients
• Two 40 microliter modulation loops with 30 second load and inject cycle for comprehensive sampling
• Flow splitting via zero dead volume T piece routing most flow to DAD and a 75 micrometer capillary to the MS source
• Diode array detection at 214 nanometers and quartz flow cell
• Agilent 6530 Q TOF with JetStream positive ionization acquiring centroid data at eight spectra per second in MS and MSMS modes
• Data processing with Agilent OpenLAB MassHunter Spectrum Mill and GC Image software for contour plotting

Key results and discussion

• Theoretical peak capacity increased from approximately 1150 to 2250 corrected units when extending the first dimension gradient from 75 to 225 minutes
• UV and MS contour plots confirmed preservation of peak selectivity when replacing phosphoric acid with formic acid for MS compatibility
• Over one hundred peptide features were identified by automated MSMS with mass accuracy better than 10 parts per million
• Intact protein contour maps revealed clear separation of major E coli proteins with deconvoluted molecular weights and observed sodium adducts consistent with SCX buffer composition

Practical applications and benefits

• High two dimensional peak capacity enables detection and quantification of low abundant proteoforms in complex digests and lysates
• Single platform workflow allows seamless switching between peptide and intact protein analyses without hardware reconfiguration
• Combined DAD and MS detection supports both quantitative UV monitoring and high confidence identification
• Sodium accumulation in the ion source spray shield can be managed by simple periodic rinsing without major maintenance interruptions

Future trends and potentials

• Integration with faster high resolution mass analyzers to expand coverage of very low abundance species and post translational modifications
• Exploration of alternative orthogonal pairs such as hydrophilic interaction chromatography coupled with ion exchange for specialized proteomic targets
• Development of inline desalting or trapping modules to minimize salt loading on the MS source and prolong maintenance intervals
• Application of the two dimensional LC platform to top down proteomics enabling detailed characterization of intact proteoforms with native modifications

Conclusion

Combining strong cation exchange and reversed phase separations in a comprehensive two dimensional LC solution coupled with UV and accurate mass detection provides robust high peak capacity workflows for both peptide and intact protein analyses in complex proteomic samples. The method delivers detailed separation quantification and confident identification in a single automated platform.

Reference

1. Francois I Sandra K Sandra P Comprehensive liquid chromatography fundamental aspects and practical considerations a review Analytica Chimica Acta 2009 64 14 31
2. Sandra K et al Highly efficient peptide separations in proteomics Part 2 bi and multidimensional liquid based separation techniques Journal of Chromatography B 2009 877 1019 1039
3. Vanhoenacker G et al Analysis of monoclonal antibody digests with the Agilent 1290 Infinity 2D LC Solution Application Note 5991 2880EN Agilent Technologies 2013
4. Vanhoenacker G et al Tryptic digest analysis using the Agilent 1290 Infinity LC System Application Note 5990 4031EN Agilent Technologies 2009