Ultra-High-Pressure RPLC Hyphenated to an LTQ-Orbitrap Velos Reveals a Linear Relation between Peak Capacity and Number of Identified Peptides

Abstract

Currently, unbiased protein identification is mostly performed by directly coupling reversed-phase liquid chromatography (RPLC) via electrospray ionization to a mass spectrometer. In contrast to the innovations in mass spectrometric instrumentation, cutting-edge technology in RPLC has generally not been well adopted. Here, we describe the effects of increased peak capacities on the number of identified proteins and peptides in complex mixtures utilizing collision-induced dissociation on an LTQ-Orbitrap Velos, providing a rationale for using advanced RPLC technology in LC-MS/MS. Using two different column lengths and gradient times between 1 and 10 h, we found a linear relation between the obtained peak capacities and the number of identified peptides. We identified on average 2516 proteins in the tryptic digest of 1 μg of HeLa lysate using an 8 h gradient on a 50 cm column packed with 2 μm C18 reversed-phase chromatographic material.

Rapid and Deep Human Proteome Analysis by Single-dimension Shotgun Proteomics

“…the number of proteins identified per unit time). "High-content" proteomics strives to identify and quantify at least 1,000 proteins per hour of LC gradient, and ultimately 10,000 proteins in 10 h (the "10k10" goal) (32).…”

Influence of NanoLC Column and Gradient Length as well as MS/MS Frequency and Sample Complexity on Shotgun Protein Identification of Marine Bacteria

“…The smallest average peak widths were observed for the 50 cm column for all gradients (minimum <0.38). While the 15 cm column yielded largest peak widths (minimum <0.51), [Köcher et al, 2011]. Corresponding calculated peak capacities were highest for the 50 cm column, ranging from 180 to 590 ( Fig.…”

“…Although the resolution power of beads <2 μm in combination with very long analytical columns (e.g., up to 2 m) has been demonstrated [MacNair et al, 1997;Shen et al, 2005;Zhou et al, 2012], the vast majority of shotgun analyses applies columns with 2 to 3 μm beads and short to intermediary column lengths (commonly 15-25 cm) [Xie et al, 2012]. Independent of the column/hardware LC setup, longer gradient times generally allow for higher peak capacities [Guiochon, 2006;Köcher et al, 2011;Liu et al, 2007]. This effect is, however, accompanied by peak broadening, and the long gradient times reduce the sample throughput of the instrument -a crucial factor in daily routine.…”

mzDB: A File Format Using Multiple Indexing Strategies for the Efficient Analysis of Large LC-MS/MS and SWATH-MS Data Sets *

“…The continuous improvement of mass spectrometers (1)(2)(3)(4) and HPLC systems (5)(6)(7)(8)(9)(10) and the rapidly increasing volumes of data they produce pose a real challenge to software developers who constantly have to adapt their tools to deal with different types and increasing sizes of raw files. Indeed, the file size of a single MS analysis evolved from a few MB to several GB in less than 10 years.…”

“…Indeed, the file size of a single MS analysis evolved from a few MB to several GB in less than 10 years. The introduction of high throughput, high mass accuracy MS analyses in data dependent acquisitions (DDA) 1 and the adoption of Data Independent Acquisition (DIA) approaches, for example, SWATH-MS (11), were significant factors in this development. The management of these huge data files is a major issue for laboratories and raw file public repositories, which need to regularly upgrade their storage solutions and capacity.…”