In shotgun proteomics it is desirable to identify and quantify a large number of individual peptides from complex samples, such as tryptic digests of human plasma samples or whole cell lysates in the shortest possible time. Complexity and concentration range, however, pose a great challenge to the MS instrumentation in terms of sensitivity, resolution and dynamic range. Several hardware modifications of a bench-top UHR-TOF instrument were carried out and evaluated addressing these particular performance aspects.

To test the impact of these modifications on proteomics performance, different complex tryptic digests (Lysates of Escherichia coli, Saccharomyces cerevisiae, human plasma) were mixed with stable isotope labeled peptides or digests of standard proteins at known concentrations, spanning a range of several orders of magnitude. Samples were analyzed with nano-flow UHPLC and a CaptiveSpray ion source connected towith nano-UHPLC MS/MS on the modified UHR-TOF. For peptide identification and quantitative analysis the MaxQuant software package was used (Nature Biotechnology 26, 1367 - 1372 (2008)).

For higher sensitivity at fast acquisition speed, ion extraction from the collision cell into the orthogonal acceleration of the TOF-analyzer was improved by using a novel collision cell design was used. Increased resolution without changing the effective flight path could be achieved with a modified reflectron. In addition, a faster detector (reduced width of individual ion signals) led to further improvements in resolving power.
Using an optimized detector digitizer combination, a threefold higher dynamic range was observed. However in complex samples, the dynamic range is also limited by the capability of the instrument to resolve nearly isobaric compounds. The performance improvements were analyzed in a label-free quantification experiment, evaluating in particular the number of quantifiable peptides over the entire dynamic range. As a defined model system for complex proteomics samples, a mixture of 48 standard proteins spanning a concentration range of five orders of magnitude (Universal Proteomics Standard, UPS-2, Sigma) was spiked into samples of 500ng E. coli, S.Cerevisiae (higher complexity) or human plasma (wider dynamic range) digests at a concentration of 1:2. During separation in with two hour gradients, the UPS-2 peptides could be quantified based on the MS full scans at levels from 500fmol down to the low attomole range. Similar analyses with background samples of higher complexity (S. cerevisiae) and wider dynamic range (human plasma) were carried out to further evaluate the benefit of the novel hardware features.