Improved SILAC method for double labeling of bacterial proteome

Highlights

• Amino acid conversion in bacteria decreases the labeling efficiency and quantification accuracy.

• Adding high concentrations of heavy and light amino acids can efficiently inhibit the complicated amino acid conversions.

• This simple and straightforward strategy facilitated the full incorporation of amino acids into the bacterial proteome.

Abstract

Stable isotope labeling with amino acids in cell culture (SILAC) is a robust proteomics method with advantages such as reproducibility and easy handling. This method is popular for the analysis of mammalian cells. However, amino acid conversion in bacteria decreases the labeling efficiency and quantification accuracy, limiting the application of SILAC in bacterial proteomics to auxotrophic bacteria or to single labeling with lysine. In this study, we found that adding high concentrations of isotope-labeled (heavy) and natural (light) amino acids into SILAC minimal medium can efficiently inhibit the complicated amino acid conversions. This simple and straightforward strategy facilitated complete incorporation of amino acids into the bacterial proteome with good accuracy. High labeling efficiency can be achieved in different bacteria by slightly modifying the supplementation of amino acids in culture media, promoting the widespread application of SILAC technique in bacterial proteomics.

Significance

Amino acid conversion in bacteria decreases labeling efficiency, limiting the application of Stable isotope labeling with amino acids in cell culture (SILAC) in bacterial proteomics to auxotrophic bacteria or single labeling with lysine. In this study, we found that high concentrations of isotope-labeled (heavy) and natural (light) amino acids facilitate full incorporation of amino acids into the bacterial proteome with good reproducibility. This improved double labeling SILAC technique using medium supplemented with high concentrations of amino acids is suitable for quantitative proteomics research on both gram-positive and -negative bacteria, facilitating the broad application of quantitative proteomics in bacterial studies.

Introduction

Bacteria are important for human life, agriculture, and industrial processes. However, many pathogenic bacteria seriously threaten human health. Although antibiotics are available to treat bacterial infections, their overuse in recent years has led to the emergence of drug-resistant strains. Quantitative proteomics is a useful method to study the virulence and resistance mechanisms of bacteria [1,2].

Quantitative proteomics methods include two-dimensional electrophoresis, chemical labeling, metabolic labeling, and label-free quantification. These methods are used in different types of analyses based on their specific advantages and limitations. Two-dimensional electrophoresis is an older method with low repeatability and limited ability to detect low-abundant, hydrophobic, and high pI proteins [3]. More recently, isobaric tags for relative and absolute quantitation (iTRAQ) [4] and tandem mass tags (TMT) [5] have become popular chemical labeling techniques for protein and peptide quantification. These methods are able to label samples of any type, with up to 10 different samples in a single analysis [6,7]. Disadvantages of chemical labeling are dynamic range compression and errors introduced during sample handling [4,5,8,9]. Label-free quantitation is simple and cost-effective but does not allow multiplexing and has insufficient accuracy in quantitation [10,11].

The most popular and accurate quantitative method in proteomic studies is stable isotope labeling with amino acids in cell culture (SILAC) [12]. In SILAC, one cell population is cultured with isotope-labeled (heavy) amino acids and a second cell population is cultured with natural (light) amino acids. The differentially treated cell samples can be mixed at the level of intact cells, namely at the very initial step of the experimental workflow to minimize experimental errors or bias resulted from separate handling procedures [13]. Thus, SILAC is particularly suitable for those approaches with extensive sample processing, such as subcellular fractionation, affinity purification of protein complex or enrichment of peptides with post-translational modifications (PTM).

SILAC has been widely used for the proteomics in Drosophila melanogaster [14], Caenorhabditis elegans [15,16], Saccharomyces cerevisiae [17], mammalian cells [18], and mice [19]. However, SILAC has been used successfully only in a small amount of bacteria including Escherichia coli [20] and Bacillus subtilis [21], Salmonella typhimurium [22], Rhodobacter sphaeroides [23], Psedomonas aeruginosa [24], Listeria monocytogenes [25],Neisseria gonorrhoeae [26]. The reason is that this method requires the use of auxotrophic strains or deletion of the lysine and arginine biosynthesis pathways in bacteria [27]. Furthermore, some auxotrophic strains of bacteria did not survive well in normal medium [28]. A recent proteomics study used wild-type (non-auxotrophic) E. coli but only achieved single lysine labeling [16,20,29], resulting in the loss of almost all quantitative information about peptides with C-terminal arginine and a dramatic decrease in the number of proteins identified. Obviously, the complicated amino-acid conversion presenting in wild-type bacteria limited the application of SILAC in bacterial proteomics [21].

To facilitate the widespread use of SILAC in bacterial proteomics, we developed a method that addresses the problems of metabolic alterations. We found that the incorporation rates of lysine and arginine and the complexity of spectra were related to amino acid concentrations in the culture medium, especially the concentrations of heavy amino acids in the minimal medium. Our results suggest that the supplement of high concentrations of heavy and light amino acids can successfully suppress amino acid interconversions and achieve double labeling of proteins in both Gram-positive and -negative bacteria.