biorxiv.org
High-performance proteomics at any chromatographic flow rate
Current applications of mass spectrometry-based proteomics range from single cell to body fluid analysis that come with very different demands regarding sensitivity or sample throughput. Additionally, the vast molecular complexity of proteomes and the massive dynamic range of protein concentrations in these biological systems require very high-performance chromatographic separations in tandem with the high speed and sensitivity afforded by mass spectrometer. In this study, we focussed on the chromatographic angle and, more specifically, systematically evaluated proteome analysis performance across a wide range of chromatographic flow rates (0.3 – 50 μL/min) and associated column diameters using a Vanquish Neo UHPLC coupled online to a Q Exactive HF-X mass spectrometer. Serial dilutions of HeLa cell line digests were used for benchmarking and total analysis time from injection-to-injection was intentionally fixed at 60 minutes (24 samples per day). The three key messages of the study are that i) all chromatographic flow rates are suitable for high-quality proteome analysis, ii) capLC (1.5 μL/min) is a very robust, sensitive and quantitative alternative to nanoLC for many applications and iii) showcased proteome, phosphoproteome and drug proteome data provide sound empirical guidance for laboratories in selecting appropriate chromatographic flow rates and column diameters for their specific application.
### Competing Interest Statement
R.Z. and C.P. are employees of Thermo Fisher Scientific. BK is a non-operational co-founder and shareholder of MSAID. The other authors declare no competing interests.
* ABBREVIATIONS
:
ACN
: Acetonitrile
CAA
: Chloroacetamide
capLC
: Capillary Liquid Chromatography
CSF
: Cerebrospinal Fluid
CV
: Coefficient of Variation
DDA
: Data-dependent Acquisition
DIA
: Data-Independent Acquisition
DMSO
: Dimethyl Sulfoxide
DTT
: Dithiothreitol
EC50
: Effective Concentration to Reduce 50% of Protein Binding to Kinobeads
FBS
: Fetal Bovine Serum
fmol
: Femtomole
FDR
: False Discovery Rate
FWHM
: Full Width at Half Maximum
HeLa
: Human Cervical Carcinoma Cell Line
HLB
: Hydrophilic-Lipophilic Balance
HPLC
: High-Performance Liquid Chromatography
i.d.
: Internal Diameter
Kdapp
: Apparent Dissociation Constant
LC
: Liquid Chromatography
LC-MS/MS
: Liquid Chromatography-Tandem Mass Spectrometry
µg
: Microgram
min
: Minute
µL
: Microliter
µLC
: Microflow Liquid Chromatography
mm
: Milimeter
mM
: Milimolar
MS
: Mass Spectrometry
ng
: Nanogram
nL
: Nanoliter
nLC
: Nanoflow Liquid Chromatography
nM
: Nanomolar
PSM
: Peptide-spectrum Match
RT
: Retention Time
SPD
: Samples per Day
SWATH-MS
: Sequential Window Acquisition of All Theoretical Mass Spectra
TFA
: Trifluoroacetic Acid
Å
: Angstrom