Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples

Abstract

Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-μm i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 μL/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of n-Dodecyl β-D-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of ~49,000–94,000 unique peptides, corresponding to ~6,700–8,300 protein groups using only 100–1000 ng of HeLa tryptic digest (equivalent to ~500–5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify ~20,000 phosphopeptides from 100 μg of MCF-7 cell lysate.

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

“…demonstrated to enable targeted quantification of the majority of epidermal growth factor receptor pathway proteins in 10-100 mammalian cells. We have also demonstrated that the addition of a MS-compatible detergent, n-Dodecyl -D-Maltoside (DDM), can significantly reduce surface adsorption for improving sample recovery 10 . Most importantly, we have recently developed a nanoPOTS (nanodroplet Processing in One Pot for Trace Samples) platform 11 to dramatically improve sample processing efficiency for small number of cells down to single cells.…”

Optimization of Data-Independent Acquisition Mass Spectrometry for Deep and Highly Sensitive Proteomic Analysis

“…The sensitivity and depth of proteomic analysis have been improved through development of mass spectrometry (MS), pretreatment, and liquid chromatography (LC) separation technologies, which have made it possible to analyze lowly expressed proteins, including those of interest [2,3,4,5]. For deep proteomic analysis, multi-dimensional protein identification technology (MudPIT), in which digested peptides are fractionated on strong cation exchange, high-pH C18, and other LC columns followed by LC-MS/MS analysis, is the primary approach used [1,6,7,8,9]. Moreover, by combining MudPIT with isobaric tags, such as tandem mass tag and isobaric tags for relative and absolute quantification, in-depth comparative proteomic analysis is possible [10,11].…”

Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality

“…Reproducible and in-depth proteomic analysis of samples smaller than 500,000 cells requires signi cant improvements in sensitivity over standard approaches. The necessary technological improvements are actively being pursued in our lab (21,(33)(34)(35)(36) and others (20,(37)(38)(39) with great success in recent years. However, much more work is needed to make these technologies available to less specialized laboratories.…”