Published: January 30th, 2019
We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).
3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect < 2.5 µm of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 μm, with a detection limit of 1.13 pg/m3.
Aerosol or airborne PM contains proteins from various biological origins, including viruses, prokaryotes, fungi, plants (pollen), and animals (insects, human)1. The ratio of protein content in PM is estimated to be nearly 5%, which has been implicated to play a major role as an airborne allergen2. Recent reports suggest that urban ambient aerosol of various sizes contain amino acids with ratios ranging between 0.5% and 2%3. In addition, chemical modifications of PM proteins have been suggested to be generated by the reactions of proteins with various pollutants, such as O3, nitrogen dioxide (NO2), and sulfur dioxide (SO2)4.
3-NT—a protein modification—is generated by the nitration of tyrosine residues. A higher concentration of O3 and NO2 has been shown to promote the nitration of protein molecules in pseudo-atmospheric space5,6. The nitration of tyrosine residues in the polypeptide chain enhances the allergenic potential of pollen proteins7.Thus, the quantification and assessment of airborne 3-NT is an important aspect in addressing the concerns of environmental health.
3-NT is also known as a biomarker of oxidative and nitrosative stress8. An emerging body of evidence has shown a significant association of 3-NT content with several human diseases9,10. Owing to its detrimental effects, the detection and quantitative estimation of 3-NT in a biological sample hold great relevance in determining the condition of an individual's health. In recent years, diverse methods have been introduced to estimate the 3-NT content, including enzyme-linked immunosorbent assays, HPLC, and LC/MS11. In previous studies, we have reported a detection method using the HPLC-ECD technique bestowed with several advantages compared to previous methods. For example, the HPLC-ECD does not require an extraction or derivatization procedure which makes it a relatively simple assay system12.We have confirmed that this method is applicable to detect 3-NT from biological samples (plasma from human and rat).
Although many investigations have emphasized the detection of 3-NT in biological samples, its detection in nonbiological samples remains elusive, and hence the present study has been pursued. In fact, to assess the harmful effect of airborne 3-NT, a quantitative method that is useful to detect 3-NT directly from airborne particles is required; therefore, we applied the existing HPLC-ECD method to detect 3-NT from PM2.5 collected on a glass-fiber filter. By using the technique, 3-NT could be detected directly from small pieces (6 mm-diameter round holes) of the sample from a PM collection filter.We further evaluated the content of 3-NT for various PM sizes and measured the detection limit of 3-NT.The present article proposes a high-sensitive and high-throughput method to detect 3-NT directly from both nonbiological and biological samples.
1. Collection of Total Suspended Particles, PM2.5, or PM7, and the Hydrolysis Method for PM Proteins
2. Determination of 3-NT Content in TSP, PM2.5, and PM7 via a High-performance Liquid Chromatography and Electrochemical Detection (HPLC-ECD) System
Note: See Figure 3.
3. Analysis of Chromatograms and Quantification of 3-NT Content in PM7
The HPLC-ECD principle is simple. Samples containing the target are separated by the HPLC column and mobile phase, and the separated target compound is reduced and/or oxidized in the electrochemical detector.
In the HPLC-ECD system, a 3-NT peak is detected in approximately 22 minutes. ECDs (PEC-510 and HTEC-500) are connected in tandem in the HPLC line. The first ECD reduces the hydroxyl groups in the compounds, and the next oxidizes the reduced chemicals. ECDs have a highly sensitive detection range of 10-15 M. The detection limit of the HPLC-ECD system was 1.13 pg/m3, which was calculated as the mean basal signal (mV) of 10 parts x 3.
Representative chromatograms are shown in Figure 4. By analyzing a pure 3-NT standard, a definitive peak with a stable baseline can be detected (Figure 4A). 3-NT in PM7 was also measured using 6 mm, round-shaped samples (Figure 4B).
Regarding the accuracy of 3-NT detection capability, when a 3-NT standard was injected in HPLC, the peak corresponding to 3-NT was detected at a retention time of 20 minutes. In PM samples, 3-NT was separated from other substances, and the independent peak was detected at the same retention time as the standard. A typical standard curve is shown in Figure 5, where linearity is observed between 5 and 40 nM. Linearity can be seen up to 500 nM (data not shown).
Table 1 shows the 3-NT contents in the atmosphere, calculated as pg/m3 air. The highest concentration of 3-NT in a PM2.5 prefilter was observed in #3 (PM7.3-4.5).
Figure 1: Collection scheme for TSP, PM7, and PM2.5. (A) The total suspended particles (TSP) are collected directly onto a collection filter (at a flow rate of 1,000 L/min). (B) When PM7 is collected, large particles are excluded by a size selector (at a flow rate of 1,000 L/min). (C) PM2.5 is collected through four prefilters (at a flow rate of 116 L/min). A quartz filter is commonly used as a backup filter for PM7 and PM2.5 or as a collection filter for TSP, as shown in this figure. Please click here to view a larger version of this figure.
Figure 2: Preparation of a 6 mm-round circle. The collection filter was cut using a hollow punch tool with a 6 mm diameter. Then, the sample was stored in a 1.5 mL microtube. Please click here to view a larger version of this figure.
Figure 3: Schematic diagram of the HPLC-ECD system. 3-NT in an injected sample was separated in the HPLC column through which mobile phase flows. The 3-NT was reduced to aminotyrosine, oxidized to iminotyrosine, and detected electrochemically in the detector. Please click here to view a larger version of this figure.
Figure 4: Typical chromatograms in the HPLC-ECD system. (A) Pure 3-NT analyzed by the HPLC-ECD system. (B) 6 mm circle sample from a PM7 filter used to detect 3-NT. A 3-NT peak is indicated by an arrow. Please click here to view a larger version of this figure.
Figure 5: Standard curve of pure 3-NT samples. The results at a lower concentration are indicated in another graph. Please click here to view a larger version of this figure.
|8.25 ± 0.37
|29.66 ± 1.94
|74.37 ± 4.09
|32.70 ± 0.75
|4.21 ± 0.91
|89.16 ± 6.36
|10.34 ± 2.09
Table 1: 3-NT in the atmosphere measured by the HPLC-ECD system. The 6 mm circular spots from each filter were measured using the HPLC-ECD system. Prefilters #1 - #4 were collected in the same period. Note that the concentrations of 3-NT in the air are influenced by various factors, such as the collected date, year, or location. The data (n = 3 - 10) are represented as the mean ± the standard error.
This article describes a quantification method to evaluate 3-NT in airborne PM collected on quartz filters using highly sensitive HPLC-ECD techniques.
In general, 3-NT measurement methods have been developed as biomarkers of oxidative stress in human diseases. Antibody-based methods (e.g., the enzyme-linked immunosorbent assay) are considered as semi-quantitative because there is no strict assay validation and it is difficult to assess the test's reliability. HPLC with electrochemical detection (ECD) and mass spectrometry-based assays have adequate sensitivity for the quantification of 3-NT13. Although they are sensitive, mass spectrometry-based methods like GC-MS or GC-MS/MS require the derivatization of amino acids, and the process of derivatization often results in the formation of artifacts14.
Compared to both the GC and LC techniques, HPLC-ECD is relatively less expensive and has a sufficient sensitivity to measure 3-NT. Additionally, the derivatization step in GC-MS and LC-MS often requires additional time. Although the method presented here requires 16 hours for the protein digestion step, it can, nevertheless, be carried out overnight (as described above, about 30 minutes of hands-on time is required per sample).Automatic repeat measurement by using an autosampler may provide a high-throughput measurement system.Previous studies have reported immunological methods to measure 3-NT in PM2,7; however, such methods could not detect 3-NT in the winter season due to the low levels of 3-NT in the PM15.
The HPLC-ECD method has additional advantages over other standard methods; for example, (i) an extraction process is not required in this method, (ii) it is completely detergent-free, (iii) it has minimal sample requirement, and, importantly, (iv) it has high sensitivity. Particles that are collected onto filters are frequently separated by sonication for further investigations, including the quantification of various components. Detergents are also used to isolate PM-bound proteins from filters. However, these additional steps increase the risk of contamination, sample loss, and underestimation due to extraction efficiency, and furthermore, most detergents are incompatible with LC/MS. In the present HPLC-ECD method, HPLC samples can easily be separated from the filter by using a simple hollow punch and without the requirement of an additional sample preparation process. The area of the round-shaped sample is 28.3 mm2, which is very small compared to the size of the original quartz filter (8 x 10 inch = 203.2 x 254 mm = 51,600 mm2).
The HPLC-ECD condition has been reviewed carefully to avoid interference to the 3-NT signal, as described previously12. A strong acidic pH of mobile phase and an adequate concentration of acetonitrile is important for detection.Using these conditions, other compounds, including nitro base, can be separated from nitrotyrosine.The present condition is suitable for the detection of 3-NT from samples with a different physical property (e.g., particulate matter and plasma).
To evaluate 3-NT in the air, the measurement of the filter weight and the elimination of the background are critical steps. In general, over approximately 100 mg of PM7 are collected over a period of four to seven days; however, several factors affect the filter weight before and after the PM7 collection, including humidity and static electrical charge. To avoid these effects, the stabilization of the filter weight for long periods is necessary under subequal temperature and humidity. The location where the electronic balance is installed should be maintained in a stable environment. For sample preparation, it is important to correct the background effects from the nonspecific protease cocktail and the ultrafiltration membrane, which often show a similar peak to 3-NT.
This method can be applied to other particles, including those in indoor environments. The nitration of proteins may increase their allergenic potential2. Therefore, this method may help to evaluate environmental cleanliness and prevent nitrosative stress.
In this study, we report a highly sensitive measurement method for atmospheric 3-NT of air sampler filters with easy-to-handle and inexpensive apparatus. The generation of 3-NT in the atmosphere is associated with environmental pollutants such as O3, NO2, PM proteins, and meteorological elements which affect human allergenicity. In conclusion, the method developed in the present investigation may help to assess the atmospheric reaction of O3, various pollutants, and PM proteins under various meteorological conditions. With these endeavors of developing HPLC-ECD for an estimation of 3-nitrotyrosine in the atmosphere, we anticipate better environmental cleanliness, improving human health.
The authors have nothing to disclose.
The authors thank Masayuki Kubo of the US Environmental Protection Agency for his help with the TSP/PM2.5 sampling. This work was supported in part by JSPS KAKENHI Grant Number JP16K15373 and JP18H03039.
|Quartz filter, backup quartz filter
|PALL life sciences
|Tissuquatrz 2500QAT-UP, 8x10in
|High-volume air sampler
|SIBATA SCIENTIFIC TECHNOLOGY
|Particle size selector for SPM
|SIBATA SCIENTIFIC TECHNOLOGY
|SPM (suspended particulate matters) is defined in Japan that particles with 10 um of 100% cut-off diameter. SPM is almost identical to PM7.
|Low-volume air sampler
|SIBATA SCIENTIFIC TECHNOLOGY
|Size classification unit for PM2.5
|SIBATA SCIENTIFIC TECHNOLOGY
|Quartz filters for classification (PM4.5–2.5, PM7.3–4.5, PM15–7.3, and TSP-PM15)
|Non-specific protease cocktail
|Pronase from Streptomyces griseus
|AMICON ULTRA 0.5 mL - 10kDa cutoff
|standalone HPLC-ECD unit (HTEC-510) and Pre-electrolysis cell (PEC-510), includes pump
|Hitachi High-Tech Science
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