Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used by researchers for separating, identifying and quantifying substances - such as proteins - in complex biological samples. There are three main types – nanoflow, microflow and standard flow – each of which is distinguished by the choice of column size and flow rate.
Nanoflow offers the ultimate in sensitivity and is usually the method of choice for proteomics. But its drawbacks, which include complexity and slowness, can limit the comprehensiveness, robustness, and throughput of these analyses, particularly when researchers are dealing with highly complex samples with wide-ranging protein concentrations - such as tissues or body fluids collected in clinical studies.
Microflow is often considered as a compromise - as it is quicker and easier to carry out than nanoflow but offers greater sensitivity than standard flow. But despite it being around since the 1970s, the approach has not been widely adopted by analytical laboratories. But recent advances in technologies and data analyses are now opening new opportunities for its revival.
In a new study, published in Nature Communications, a team of researchers set out to evaluate the merits of carrying out online microflow LC-MS/MS for quantitative discovery proteome analysis.1
The researchers prepared more than 2,000 samples for analysis - from human cell lines, tissues and body fluids. They then performed LC-MS/MS at a flow rate of 50µl/minute using standard high-performance liquid chromatography (HPLC) equipment and a commercial analytical reversed-phase chromatography column (150mm inner diameter) - coupled online to a sensitive and rapid online mass spectrometer.
During these experiments, the team used ultrapure water generated from an ELGA PURELAB® laboratory water purification system to minimize the risk of adding contaminants that may impact on their results.
Using deep proteome analysis, the researchers identified more than 9,000 proteins and more than 120,000 peptides in 16 hours. By multiplexing samples using tandem mass tags, they achieved further increases in throughput to 11 proteomes.
The team also demonstrated that the system could identify >30,000 phosphopeptides in 12 hours and analyse protein-protein or protein-drug interaction experiments in 20 minutes per sample. They also showed that they could use the same column to analyse >7,500 samples without any apparent loss of performance.
These results show that most of the limitations of nanoflow can be overcome using microflow LC-MS/MS at a very moderate loss of practical sensitivity. The approach offers marked improvements in robustness, throughput and reproducibility of quantification and is suitable for a broad range of proteomics applications.
This system could be game-changing for the field of proteomics - due to the ease of its technical implementation, the wide range of feasible applications and the very high-quality data that makes it suitable for the analysis of clinical samples.
ELGA’s expert engineers, chemists and scientists are at the forefront of technological innovation. We continue to introduce game-changing features to the laboratory water market.
Dr Alison Halliday
After completing an undergraduate degree in Biochemistry & Genetics at Sheffield University, Alison was awarded a PhD in Human Molecular Genetics at the University of Newcastle. She carried out five years as a Senior Postdoctoral Research Fellow at UCL, investigating the genes involved in childhood obesity syndrome. Moving into science communications, she spent ten years at Cancer Research UK engaging the public about the charity’s work. She now specialises in writing about research across the life sciences, medicine and health.