Here, we describe an in-house built ultra-high pressure liquid chromatography 
(UHPLC) system, with little complexity in design and high separation power 
combined with convenience in operation. This system enables the use of long 
columns of 40 cm packed with 1.8 μm particles generating pressures below 1000 
bar. Furthermore, the system could be operated at flow rates between 50 and 200 
nL min(-1) while maintaining its separation power. Several gradients were 
optimized ranging from 23 to 458 minutes. With the longest gradient we 
identified over 4500 protein groups and more than 26,000 unique peptides from 1 
μg of a human cancer cell lysate in a single run using an Orbitrap Velos - a 
level of performance often seen solely using multidimensional separation 
strategies. Further experiments using a mass spectrometer with faster sequencing 
speeds, like the TripleTOF 5600, enabled us to identify over 1400 protein groups 
in a 23 min gradient. The TripleTOF 5600 performed especially well, compared to 
the Orbitrap Velos, for the shorter gradients used. Our data demonstrate that 
the combination of UHPLC with high resolution mass spectrometry at increased 
sequencing speeds enables extensive proteome analysis in single runs.