Increased MS Protein Identification Rates Using 75 cm Long nano LC C18 Separation Columns: Pushing the Limits of Bottom-Up Proteomics

[Orbitrap_SciLib]

Daniel Lopez-Ferrer1, Michael Blank1, Stephan Meding2, Aran Paulus1, Romain Huguet1, Remco Swart2, Andreas FR Huhmer1

Introduction: Bottom-up proteomics has always aimed to identify and quantify the complete proteome from a cell, tissue, or whole organism. Many advances have been made in the last 15 years. Still sample separation is one of the technological challenges. Separation columns have continuously increased in length. So far, 50 cm columns were the longest commercially available high performance nano LC columns. It was evaluated whether using newly available 75 cm separation columns will significantly increase peptide and protein identification rates. Methods: A Thermo Scientific™ EASY-nLC™ 1200 LC system with a Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer were used to analyze a HeLa cell lysate with a 75 cm long 75 µm ID Thermo Scientific™ Acclaim™ PepMap™ capillary column using both 2 and 4 hour gradients. The results were compared with those obtained under the same conditions with a 50 cm column, which was until now the longest commercially available high performance nano LC column for bottom-up proteomics. In both cases, the columns were used in EASY-Spray™ column format. Results: The length increase resulted in the separation and detection of 10% more unique peptides, and 7% more protein identification in a 4 hour gradient, with protein identifications exceeding 5700 proteins for a single injection of mammalian cell lysate. More importantly, longer columns showed better reproducibility as seen by increased correlation among technical replicates, higher numbers of quantifiable peptides, and a smaller coefficient of variance (CV), resulting in improved protein quantification for complex lysates by high resolution accurate mass (HRAM) LC-MS. Novel aspect: New levels of separation efficiency and as a result of peptide identification were achieved by using 75 cm long separation columns.


1Thermo Fisher Scientific, San Jose, USA 2Thermo Fisher Scientific, Germering, Germany