Particularly for targeted quantitative MS assays such as selected reaction monitoring (SRM) 8 or, more recently, parallel reaction monitoring (PRM) 9, where the mass spectrometer is focused on a small number of analytes to maximize sensitivity and quantitative accuracy/precision 10, standard analytical HPLC columns (2.1 mm inner diameter, ID) are frequently used 11, 12, 13, 14, 15 to address the aforementioned challenges. These factors can limit the reproducibility of peptide identification and quantification as well as the comprehensiveness, robustness and throughput of proteome analysis, particularly when analyzing samples of high complexity or wide dynamic range of protein concentrations as represented by tissues and body fluids 7. However, this comes at the cost of the challenge of manufacturing reproducible and long-lasting columns, maintaining stable ESI over extended periods of time, rapid chromatographic overloading, mass spectrometric saturation and often long, unproductive overhead times for sample transfer at low flow rates 4, 5, 6. Nano-flow liquid chromatography (nano-flow LC) has been the mainstay in proteome research for >20 years 1, primarily because low flow rates improve peptide ionization by electrospray (ESI) for mass spectrometry (MS) and, hence, sensitivity 2, 3.
This study demonstrates that micro-flow LC–MS/MS is suitable for a broad range of proteomic applications. We show that the same column can be used to analyze >7500 samples without apparent loss of performance.
The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. Here we show that micro-flow LC–MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (2000 samples of human cell lines, tissues and body fluids.
Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC–MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness.
0 kommentar(er)
