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.
Highlights
Proteins regulate and catalyze all cellular processes
This study presents the development of a sample preparation method, which overcomes the limitations in lysis and extraction of in-solution digestion (ISD) but preserves its straight-forward approach
Comprehensive, accurate and deep proteome analysis relies on the reproducible generation of peptides from the entity of proteins of a given sample type
Summary
Proteins regulate and catalyze all cellular processes. The analysis of the entity of proteins is of utmost importance for a molecular understanding of life. Technical progress in the fields of mass spectrometry (MS) and bioinformatics has enabled the realization of deep and reproducible proteomic studies at increased throughput 1. The majority of these studies are currently performed using a bottom-up approach, which relies on the digestion of proteins into smaller peptides 2. E.g. sodium dodecyl sulfate (SDS), or chaotropic agents, such as urea, for protein extraction and support sample lysis by physical disruption methods, such as heat, ultra-sonication or grinding [3,4,5]. Enhancing lysis by use of physical disruption methods and subsequent protein purification requires additional sample handling steps, which are associated with attendant losses, biases and possible contaminations, while being time-consuming and labor-intensive
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