Determination of Staphylococcal Phenol-Soluble Modulins (PSMs) by a High-Resolution HPLC-QTOF System Determination of Staphylococcal Phenol-Soluble Modulins (PSMs) by a High-Resolution HPLC-QTOF System

Phenol‐soluble modulins (PSMs) are multifunctional, amphipathic, α‐helical peptides produced by virtually all staphylococcal strains. They have recently drawn much atten‐ tion owing to the key contribution of some PSM peptides to staphylococcal virulence, in particular in highly virulent Staphylococcus aureus. High concentration of PSMs may cause cytolysis, damaging neutrophils, immune cells, red blood cells and white blood cells. Furthermore, all PSMs contribute to biofilm structuring and the dissemination of biofilm‐associated infection. Here we describe a method for PSM analysis in S. aureus by high‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry system (HPLC‐QTOF).


Introduction
Phenol-soluble modulins (PSMs) are a family of multifunctional, amphipathic and α-helical peptides produced by virtually all staphylococcal strains [1][2][3]. They have recently drawn much attention owing to the key contribution of some PSM peptides to staphylococcal virulence, in particular in highly virulent Staphylococcus aureus [4]. In addition, they have antibacterial activity, likely to compete with other environmental rival bacteria such as streptococci [5,6], as well as biofilm structuring and dissemination functions [7,8]. Staphylococcus aureus is an important and versatile opportunistic human pathogen that can cause a wide range of acute and chronic diseases, which range from superficial infections to invasive and life-threatening ones [9,10].
PSMs were first identified in 1999 by the group of Seymour Klebanoff with the description of a "pro-inflammatory complex". They were isolated by hot phenol extraction from S. epidermidis culture filtrate [11]. The peptides were named PSMα, PSMβ and δ-toxin. Afterwards, the PSM compositon of S.aureus was also analyzed more systematically, which include the shorter α-type (four PSMα1-PSMα4 peptides and δ-toxin) and the longer β-type (the PSMβ1 and PSMβ2 peptides). The PSMs are encoded at three different locations in the genome. Four PSMα1-PSMα4 peptides are encoded in the PSMα operon. PSMβ1 and PSMβ2 are encoded in the PSMβ operon. δ-toxin is encoded within the coding sequence for RNAIII [12,13].
S. aureus are considered to be second to salmonella as important foodborne pathogens, which have been frequently reported as agents leading to outbreaks of diseases caused by enterotoxins in ready-to-eat food and food products. The pathogenic mechanisms of S. aureus have been examined extensively through different stages of infection. Various staphylococcal components contribute to virulence with an improved understanding of specific functions [14][15][16].Proteomics studies have revealed that the production of virulence factors by different isolates of S. aureus is diverse and only a few of these seem to be invariantly produced [17]. PSMs are the most commonly identified staphylococcal virulence factors, especially in the community-associated (CA)-MRSA lineages. Recently, it was found that many in vitro PSM phenotypes were strongly inhibited by serum lipoproteins, and can exert their contribution to pathogenesis by intracellular killing and participate in neutrophil killing after phagocytosis [18]. In addition, PSMs at low concentration can lead to immune cell chemotaxis and inflammatory reaction. High concentration of PSMs may cause cytolysis, damaging neutrophils, immune cells, red blood cells and white blood cells [19,20]. Thus, it is important to develop a rapid, specific and accurate method to detect PSMs in food products for the prevention of transmission.
At present, there are only a few methods for determination of PSMs, such as imaging mass spectrometry and liquid chromatography-ion trap or quadrupole mass spectrometry [11,21]. The sample pre-treatment of the method using imaging mass spectrometry is quite cumbersome and time-consuming. The resolution of the method by liquid chromatography-ion trap or quadrupole mass spectrometry is too low to separate interfering substances with similar charge-to-mass ratios. Here, we describe a simple and effective method with higher sensitivity and selectivity for PSM analysis in S. aureus by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry system (HPLC-QTOF).

Extraction of PSMs from TSB and beverage for quantitative analysis
1. The Staphylococcus aureus cells were removed by centrifugation (5000 rpm, 10 min, 4°C) after overnight growth (~16 h) in 4 mL of culture medium (TSB or beverage) at 37°C with shaking at 200 rpm. The "beverage" means pure juice or juice beverage. Soda, water and coffee were not included.

Results and discussion
Examples of preset m/z ratios and retention times of S. aureus PSMs for qualitative and quantitative analyses are shown in Table 1. Retention times may vary according to the LC system and MS detector. (d), (t) and (q) stand for doubly, triply and quadruply charged ions, respectively.
The chromatograms of PSMs extracted from the culture are shown in Figure 1. The result indicates that the new method has a high selectivity. Good linearity of the PSMs was achieved in the range 0.5~100 µg/L (R 2 > 0.99). Compared with other analytical methods, the pre-treatment of the new method is simple and rapid, and the high resolution makes the method highly sensitive and selective. The method has been used successfully for the determination of PSMs extracted from different culture mediums, such as milk, beverage, vegetable and meat.