The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

Abstract

The combination of qualitative analysis with label-free quantification has greatly facilitated the throughput and flexibility of novel proteomic techniques. However, such methods rely heavily on robust and reproducible sample preparation procedures. Here, we benchmark a selection of in gel, on filter, and in solution digestion workflows for their application in label-free proteomics. Each procedure was associated with differing advantages and disadvantages. The in gel methods interrogated were cost effective, but were limited in throughput and digest efficiency. Filter-aided sample preparations facilitated reasonable processing times and yielded a balanced representation of membrane proteins, but led to a high signal variation in quantification experiments. Two in solution digest protocols, however, gave optimal performance for label-free proteomics. A protocol based on the detergent RapiGest led to the highest number of detected proteins at second-best signal stability, while a protocol based on acetonitrile-digestion, RapidACN, scored best in throughput and signal stability but came second in protein identification. In addition, we compared label-free data dependent (DDA) and data independent (SWATH) acquisition on a TripleTOF 5600 instrument. While largely similar in protein detection, SWATH outperformed DDA in quantification, reducing signal variation and markedly increasing the number of precisely quantified peptides.

Discovery and Targeted Proteomics on Cutaneous Biopsies Infected by Borrelia to Investigate Lyme Disease*

“…It is hydrolyzed at low pH and thus minimizes interference with reverse-phase columns and mass spectrometry. It has been successfully tested in several studies, showing good performance for membrane proteins (50). Overall, we tested four protocols with MS-compatible detergents and two SDS removal protocols.…”

Precise label-free quantitative proteomes in high-throughput by microLC and data-independent SWATH acquisition

“…Cells were harvested by centrifugation and snapfrozen in aliquots equaling 10 OD. Sample preparation was performed based 31 with the following modifications. Cells were broken by bead shaking with 200 µl 0.05 M ammonium bicarbonate in a FastPrep instrument (3x 30 s, 6.5 m/s, 4°C), and cell pellet after centrifugation was reextracted with 200 µl lysis buffer (0.1 M NaOH, 0.05 M EDTA, 2 % SDS, 2 % 2mercaptoethanol) for 10 min at 90°C, and again for 10 min at 90°C after addition of 0.1 M acetic acid.…”

The mechanisms of extracellular electron transfer

“…Consequently, these shortfalls can limit the outcomes of proteomic investigations of electroactive biofilms in BESs. SWATH-MS is a recently developed approach that provides extensive label-free quantitation of the measurable peptide ions in a sample [116]. The approach rapidly acquires high resolution Q-TOF mass spectrometer data through repeated analysis of sequential isolation windows (swaths) throughout the chromatographic elution range [117].…”

“…Differential protein abundance from anodic S. oneidensis biofilms grown at different potentials was performed using the recently developed proteomic method SWATH-MS [116]. This approach is particularly useful for investigative analysis of low biomass samples, where quantitative proteomics is challenging.…”

“…This study uses both electrochemical and the recently developed proteomic method of SWATH-MS [116] to understand the details of EET by identifying the molecular mechanisms employed by biofilms of Shewanella oneidensis MR-1 at different applied potentials. The recently developed SWATH-MS proteomic method, has already been successfully used for quantitative proteomic analysis of anode grown S. oneidensis biofilms [27].…”