Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics Based on Acid Extraction

Abstract

The main challenge of bottom-up proteomic sample preparation is to extract proteomes in a manner that enables efficient protein digestion for subsequent mass spectrometric analysis. Today's sample preparation strategies are commonly conceptualized around the removal of detergents, which are essential for extraction but strongly interfere with digestion and LC-MS. These multi-step preparations contribute to a lack of reproducibility as they are prone to losses, biases and contaminations, while being time-consuming and labor-intensive. We report a detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which consists of three mandatory steps, acidification, neutralization and digestion. SPEED is a universal method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction by complete sample dissolution. The protocol is highly reproducible, virtually loss-less, enables very rapid sample processing and is superior to the detergent/chaotropic agent-based methods FASP, ISD-Urea and SP3 for quantitative proteomics. SPEED holds the potential to dramatically simplify and standardize sample preparation while improving the depth of proteome coverage especially for challenging samples.

Perspective on Proteomics for Virus Detection in Clinical Samples

“…A novel sample preparation technique called SPEED uses pure trifluoroacetic acid (TFA) for lysis, making detergent removal redundant, which streamlines the sample processing. 53 Moreover, for some viruses, inactivation can be achieved by heating the sample prior to MS sample preparation, for example, to 65 °C for 30 min for the inactivation of SARS-CoV-2. 12 However, there is no study that has systematically investigated virus inactivation by proteomics sample preparation methods, which is why this has to be evaluated in the future.…”

Identification of Microorganisms by Liquid Chromatography-Mass Spectrometry (LC-MS1) and in silico Peptide Mass Data

“…SPEED protocol-based preparation of microbial cells was carried as outlined in (Doellinger et al, 2018). Briefly, bacterial cells were suspended in trifluoroacetic acid (TFA, Uvasol® for spectroscopy, Merck, Darmstadt, Germany) in a sample/TFA ratio of 1:4 (v/v) and heated at 70°C for 3 min.…”

“…In this paper, two different methods of microbial sample preparation, STrap (Zougman et al, 2014) and SPEED (Doellinger et al, 2018), were utilized. Although the relatively low number of tested samples does not allow a statistically valid comparison, the tests provide some preliminary indications with regard to the influence of sample preparation on the accuracy of identification.…”

“…MS 1 peak data were acquired from a culture of Enterococcus faecalis DSM 20371; sample preparation has been carried according to the SPEED sample preparation protocol. (Doellinger et al, 2018) Top row: histogram bar chart of log 10 scaled MS 1 peak intensities (left) and the molecular weight (MW) distribution (right) of peaks after feature detection by the Minora algorithm (=original* data, blue bars) and after pre-processing and feature selection by readlcmstxtfile (processed data, red bars).…”

“…Identification results of a microbial LC-MS 1 test data set comprising 19 different strains from 11 genera and 15 species. Samples #1-#13 were prepared by the SPEED proteomic sample preparation protocol developed at RKI (Doellinger et al, 2018). The STrap (Zougman et al, 2014) method was utilized for preparing proteomic samples from strains listed at lines 14-21.…”