A new parallel high-pressure packing system enables rapid multiplexed production of capillary columns

Reversed-phase high performance liquid chromatography (HPLC) is the most commonly applied peptide separation technique in mass spectrometry (MS)-based proteomics. Particle-packed capillary columns are predominantly used in nano-flow HPLC systems. Despite being the broadly applied standard for many years capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column production with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as driving fluid, our system replaces the traditional setup of helium pressured packing bombs. By using 10x multiplexing, we have reduced the production time to just under 2 minutes for several 50 cm columns with 1.9 µm particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters (ID) and length packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station.One sentence summaryA newly constructed parallel high-pressure packing system enables the rapid multiplexed production of capillary columns.Abstract Figure

A Review: Protein Identification by LC-MS: Principles, Instrumentation, and Applications

This review will focus on protein and peptide separation studies of the period 1995 to 2010. Peptide and protein analysis have developed dramatically after applying mass spectrometry (MS) technology and other related techniques, such as two-dimensional liquid chromatography and two-dimensional gel electrophoresis. Mass spectrometry involves measurements of mass-to-charge ratios of the ionized sample. High-performance liquid chromatography (HPLC) is an important technique that is usually applied before MS is conducted due to its efficient separation. Characterization of proteins provides a foundation for the fundamental understanding of biology aspects. In this review, instrumentation, principle, applications, developments, and accuracy of the measurements of mass spectrometry will be reviewed and discussed. In addition, the principles of HPLC technology will be explained, which is necessary before applying MS.

Optimization of data-independent acquisition using predicted libraries for deep and accurate proteome profiling

ABSTRACTIn silico spectral library prediction of all possible peptides from whole organisms has a great potential for improving proteome profiling by data-independent acquisition (DIA) and extending its scope of application. In combination with other recent improvements in the field of mass spectrometry (MS)-based proteomics, including sample preparation, peptide separation and data analysis, we aimed to uncover the full potential of such an advanced DIA strategy by optimization of the data acquisition. The results demonstrate that the combination of high-quality in silico libraries, reproducible and high-resolution peptide separation using micro-pillar array columns as well as neural network supported data analysis enables the use of long MS scan cycles without impairing the quantification performance. The study demonstrates that mean coefficient of variations of 4 % were obtained even at only 1.5 data points per peak (full width at half maximum) across different gradient lengths, which in turn improved proteome coverage up to more than 8000 proteins from HeLa cells using empirically-corrected libraries and more than 7000 proteins using a whole human in silico predicted library. These data were obtained using a Q Exactive orbitrap mass spectrometer with moderate scanning speed (12 Hz) and perform very well in comparison to recent studies using more advanced MS instruments, which underline the high potential of this optimization strategy for various applications in clinical proteomics, microbiology and molecular biology.

Boosting basic-peptide separation through dynamic electrostatic-repulsion reversed-phase (d-ERRP) liquid chromatography

A simple and effective chromatographic method has allowed unprecedented resolution of basic peptide and their related impurities, including the very challenging epimeric isobaric ones.