The authors would acknowledge Scott Howard and Abigail Marinack for general support and coordination of this work.

All studies involving human urine samples were conducted adherence to the procedure approved by Pharmasan/Neuroscience Institutional Review Board (IRB).
1. Urine Sample Collection and Creatinine (Cr) Determination
<ol>
	<li>Collect 5 mL of the next morning urine samples after ca. 10 h overnight fasting in a 5 mL transport tube A containing 250 &#181;L of 3 N HCl as preservative and store at -20 &#176;C until use.</li>
	<li>Thaw and vortex 5 mL transport urine A tube and centrifuge in a centrifuge (e.g. Sorvall) (2297 x g, 10 min).</li>
	<li>Aliquot 1 mL of urine twice from the 5 mL transport tube A.</li>
	<li>Determine Cr by a urinary creatinine method26.</li>
</ol>
2. Preparation of Standard, IS and Quality Control (QC) Samples
<ol>
	<li>Prepare a stock solution of 3-NT at 100 &#181;g/mL in water with 0.01% HCOOH mobile phase A (MA) and store at -20 &#176;C.</li>
	<li>Make a 3-NT working standard solution at concentration of 100 ng/mL by diluting the 100 &#181;g/mL 3-NT stock solution with MA.</li>
	<li>Generate standards ranging from 5 to 2500 pg/mL by dilution of the 100 ng/mL working standard with 0.15 M HCl acidified blank urine free of 3-NT along with blank and double blank samples.</li>
	<li>Prepare a stock solution of IS 13C9-3-NT at 100 &#181;g/mL in water with MA and store at -20 &#176;C.</li>
	<li>Make an IS working solution at 500 pg/mL by dilution of the 100 &#181;g/mL 13C9-3-NT IS stock solution with MA.</li>
	<li>Establish five levels of QC samples covering the LLOQ, low, medium and high levels (i.e., 10, 25, 100, 500 and 1,250 pg/mL), by dilution of 3-NT working standard in the acidified blank urine.</li>
</ol>
3. Solid Phase Extraction Procedure
<ol>
	<li>Thaw and vortex urine samples, standards and QC samples.</li>
	<li>Add urine samples, standards and QC samples (250 &#181;L each) to 32&#160;wells of a clean 2 mL 96-well collection plate.</li>
	<li>Introduce the 500 pg/mL IS working solution (50 &#181;L) to each well except double blank sample well. Add 0.01% HCOOH&#160;(50 &#181;L) to the double blank sample well.</li>
	<li>Add LC-MS/MS water with 0.1% HCOOH (250 &#181;L).</li>
	<li>Mix the above mixture with an 8-channel pipette three times.</li>
	<li>Cover the plate until SPE loading.</li>
	<li>Place an MCX 96-well extraction plate and a collection reservoir on a positive pressure processor.</li>
	<li>Condition the extraction plate with flowing MeOH (200 &#181;L) through the sorbent.</li>
	<li>Equilibrate by flowing water with 2% HCOOH (200 &#181;L) through the sorbent.</li>
	<li>Load the entire volume of each of the pre-mixed samples onto the pre-conditioned extraction plate carefully with an 8-channel pipette.</li>
	<li>Set low positive pressure (e.g., 3 psi) on the positive pressure processor to allow the mixture to flow through the sorbent slowly, adjust the pressure if needed.</li>
	<li>Wash the wells by flowing water with 2% HCOOH (200 &#181;L) through the sorbent.</li>
	<li>Wash the wells by flowing methanol (200 &#181;L) through the sorbent.</li>
	<li>Wash the wells by flowing water (200 &#181;L) through the sorbent.</li>
	<li>Dry the wells completely with high positive pressure setting (e.g., 40 psi) on the positive pressure processor.</li>
	<li>Replace the reservoir with a clean 2 mL 96-well collection plate.</li>
	<li>Apply 25 mM NH4OAc at pH 9 (50 &#181;L) to elute the retained analyte and IS from the extraction plate.</li>
	<li>Pipette LC-MS water with 5% HCOOH (50 &#181;L) to neutralize the eluate.</li>
	<li>Mix with the 8-channel pipette three times and submit to LC-MS/MS station for analysis.</li>
</ol>
4. LC-MS/MS Analysis
<ol>
	<li>Accurately measure 2,000 mL of LC-MS water with a graduated cylinder and transfer it into a 2 L bottle.</li>
	<li>Pipette 200 &#181;L pure HCOOH into the above bottle containing LC-MS water.</li>
	<li>Mix thoroughly and label as mobile phase A (MA). Include initials, preparation date and expiration date.</li>
	<li>Take a 2 L bottle of LC-MS methanol and label as mobile phase B (MB) with starting date and expiration date.</li>
	<li>Set temperature of auto-sampler to 4 &#176;C.</li>
	<li>Place the collection plate with prepared samples into the autosampler.</li>
	<li>Place a PFP column (150 mm x 2.1 mm i.d., 3 &#181;m) and guard column in the oven.</li>
	<li>Set the oven temperature to 30 &#176;C.</li>
	<li>Equilibrate 10 min using the acquisition method with the LC gradient elution as shown in Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time (min)</td>
			<td>Module</td>
			<td>Events</td>
			<td>Parameter</td>
		</tr>
		<tr>
			<td>0</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>5</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>20</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>50</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>80</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>90</td>
		</tr>
		<tr>
			<td>4.01</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>95</td>
		</tr>
		<tr>
			<td>5.5</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>95</td>
		</tr>
		<tr>
			<td>5.6</td>
			<td>Pumps</td>
			<td>Pump B Conc.</td>
			<td>5</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Controller</td>
			<td>Stop</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Liquid Chromatography Gradient Elution Conditions
<ol start="10">
	<li>Create a batch list including standards, QC and urine samples.</li>
	<li>Start the batch by injection of the prepared samples (12 &#181;L).</li>
</ol>
5. Peak Identification, Integration and Data Process
<ol>
	<li>Control data acquisition and processing using the software.</li>
	<li>Identify and integrate 3-NT and IS peaks for all the samples.</li>
	<li>Establish a standard curve with the range of 10-2,500 pg/mL for 3-NT quantitation by linear regression of the peak area ratio of 3-NT and IS versus the nominal 3-NT concentration with a weighting factor of 1/x.</li>
	<li>Quantify all the samples using the standard curve.</li>
	<li>Determine if the QC samples fall in the established range.</li>
	<li>Convert the detected concentrations of urine samples to final results in the unit of nM or nmol/mmol Cr.</li>
</ol>

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The authors declare that they have no conflict of interest.

Substantial variations in concentrations previously reported in the literature for the endogenous free 3-NT in human urine samples reveal methodological problems associated with available assays8,9,10,11. Accurate determination of the low basal level of 3-NT in human urine remains a challenging task that requires special precautions for sample preparation and LC-MS/MS analysis. This protocol ou...

The effects of oxidative stress on clinical presentation have been thrust into the forefront in recent years1. One of the biomarkers being explored is 3-nitrotyrosine (3-NT), an end stable product formed when reactive nitrogen species (RNS) interact with tyrosine, a catecholamine neurotransmitter precursor. While 3-NT may have clinical value as a biomarker for RNS in vivo, the substantial changes of the properties and functions of tyrosine may adversely affect corresponding proteins and cellular functions1,2. Emerging research has suggested that 3-NT may play an important role in inflammatory conditions3, neurodegenerative disorders4,5, cardiovascular disease6 and diabetes7 as well as conditions related to oxidative stress. However, these observations are based on results from methodologies lacking in sensitivity and/or selectivity8,9,10,11. The enormous 3-NT concentration ranges for the biological samples previously reported in the literature reveal that serious analytical problems are associated with these assays and technical improvement is needed to accurately quantify the levels of 3-NT and verify its role in the pathology of these conditions.
The quantitation of free 3-NT in biological matrices presents a special challenge to man and instrument8,9,10,11. First, the trace level of endogenous 3-NT demands an ultra-sensitive detection; second, the existence of numerous structurally similar analogues, especially tyrosine, which is present in vast excess, requires a high degree of selectivity; third, the artefactual formation of 3-NT by tyrosine nitration with ubiquitous nitrate and nitrite requires special consideration during sample preparation to avoid false overestimation of 3-NT.
Among a wide variety of methodologies employed to measure 3-NT, MS/MS has been considered the gold standard method due to its superior sensitivity and selectivity11,12,13,14. Gas chromatography (GC) coupled MS/MS offers the best sensitivity, however, the indispensable sample derivatization steps are too tedious and time-consuming to be efficient for clinical utility15,16. LC-MS/MS does not require complex sample derivatization, making it the more promising option. Nonetheless, there are several obstacles to overcome such as the sensitivity of LC-MS/MS methods reported in the literature needs to improve for the measurement of low abundant 3-NT7,17,18 and the relatively long turnaround time must be shortened for high-throughput applications12,13,17,19.
Additionally, when considering clinical applications, the biological matrix used plays a significant role. It should be easy and inexpensive to obtain and non-invasive if possible20,21,22. Plasma, the traditionally used sample in the literature, is not a clinically desirable matrix, so a methodology utilizing urine which is non-invasive and cost-effective, was sought.
Several attempts to develop reliable and specific LC-MS/MS methodologies have been made using urine9,10,11. However, they have all fallen short of being either selective, reliable or efficient enough for clinical use. The effectiveness of the predominant SPE using traditional reversed-phase cartridge (C18 type) as sample cleanup for the 3-NT analysis has been questioned and a sequential SPE of strong cation exchange (SCX) and reversed phase C18-OH has been proposed6,7,19. One recently developed LC-MS/MS method utilized a multi-step purification process of manual C18 SPE, preparative high pressure liquid chromatography (HPLC), and online SPE for analysis of 3-NT23. Although this method was sensitive enough for clinical purposes, with an LLOQ of 0.041 nM, the cleanup process was intensive and tedious and required 3 mL of urine, limiting its feasibility for high-throughput. A molecularly imprinted polymer was employed as the SPE sorbent to improve the efficiency of the cleanup process14, but the resulting LLOQ (0.7 &#181;g/mL) was not low enough for clinical specimens. Another method required two-dimensional (2D) LC-MS/MS and immunoaffinity chromatography for sample cleanup in order to achieve a limit of detection (LOD) of 0.022 nM24. While all these methods have made advancements in the assessment of 3-NT, none have achieved the sensitivity, reliability, and efficiency necessary for clinical applications.
In order to investigate the pathology of free 3-NT and its role as a biomarker of oxidative stress in clinical settings, we have developed a methodology that is simple, efficient, accurate and precise, enabling for high-throughput clinical applications25. A miniaturized mixed-mode cation exchange (MCX) 96-well extraction microplate was implemented to achieve simple and effective sample cleanup and enrichment of 3-NT in a single extraction bypassing the drawbacks seen in the existing methods that require derivatization, evaporation and 2D-LC. Liquid chromatography with 0.01% HCOOH as an additive in mobile phase offered an enhanced signal response with a rapid cycle time. Selectivity was further improved through application of a mild NH4OAc elution solution for selective elution of 3-NT, and use of MRM transition for both 3-NT and the internal standard (IS). The matrix effect was compensated for by using a reduced amount of a preferred 13C-labeled isotopic IS for quantification. With the advent of this methodology, researchers and clinicians will be able to verify the role of 3-NT in clinical conditions and further explore the impact of oxidative stress.

<table><tbody><tr><td>3-Nitro-L-tyrosine</td><td>Sigma</td><td>N7389-5g</td><td></td></tr><tr><td>3-Nitro-L-tyrosine-13C&lt;sub&gt;9&lt;/sub&gt;</td><td>Sigma</td><td>652296-5.0mg</td><td></td></tr><tr><td>Mass Spec Gold Urine</td><td>Golden West Biologicals</td><td>MSG 5000-1L</td><td></td></tr><tr><td>Oasis MCX 96-well &amp;micro;Elution plate</td><td>Waters</td><td>186001830BA</td><td></td></tr><tr><td>2 mL 96 well collection plate</td><td>Phenomenex&amp;nbsp;&amp;nbsp;</td><td>AH0-7194</td><td></td></tr><tr><td>96 positive processor</td><td>Waters&amp;nbsp;</td><td>186005521</td><td></td></tr><tr><td>LC-MS Ultra CHROMASOLV methanol&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>14262-2L</td><td></td></tr><tr><td>LC-MS Ultra CHROMASOLV water</td><td>Sigma</td><td>14263-2L</td><td></td></tr><tr><td>Formic acid for mass spectrometry</td><td>Sigma</td><td>94318-50ML-F</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma</td><td>338818-1L</td><td></td></tr><tr><td>Ultra PFP propyl columns</td><td>Restek</td><td>9179362</td><td></td></tr><tr><td>5500 Triple quad</td><td>AB Sciex&amp;nbsp;</td><td>/</td><td>Contact manufacture for more detail</td></tr><tr><td>UFLC-XR</td><td>Shimadzu&amp;nbsp;</td><td>/</td><td>Contact manufacture for more detail</td></tr><tr><td>Integra 400 Plus&amp;nbsp;</td><td>Roche</td><td>/</td><td>Urinary Creatinine Jaff&amp;eacute; Gen 2 method</td></tr><tr><td>LCMS certified 12 x 32 mm screw neck vial</td><td>Waters</td><td>600000751CV</td><td></td></tr><tr><td>LCGC certified 12 x 32 mm screw neck total recovery vial</td><td>Waters</td><td>186000384C</td><td></td></tr><tr><td>5 mL transport tube</td><td>Phenix</td><td>TT-3205</td><td></td></tr><tr><td>50 mL Centrifuge tube</td><td>Crystalgen&amp;nbsp;</td><td>23-2263</td><td></td></tr><tr><td>15 mL Centrifuge tube</td><td>Crystalgen&amp;nbsp;</td><td>23-2266</td><td></td></tr><tr><td>eLine electronic pipette</td><td>Sartorius</td><td>730391</td><td></td></tr><tr><td>Microfuge centrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>A46474</td><td></td></tr><tr><td>OHAUS balance&amp;nbsp;&amp;nbsp;</td><td>Kennedy Scales, inc.</td><td>735</td><td></td></tr><tr><td>Vortex mixer&amp;nbsp;</td><td>Bernstead Thermolyne</td><td>M16715</td><td></td></tr></tbody></table>

integration of miniaturized solid phase extraction and lc-ms/ms detection of 3-nitrotyrosine in human urine for clinical applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

Figure 1 illustrates that 3-NT is completely chromatographically separated from other structurally similar tyrosine analogues under the optimized LC condition, which eliminates the co-eluting interferences due to these vastly excessive compounds and consequently enhances the degree of assay selectivity. In addition, the gradient elution with 0.01% HCOOH as additive in MA and methanol at a flow rate of 0.45 mL/min allows rapid elution of 3-NT (i.e., 3...

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Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications | Chemistry | Video

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

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9.3K Views.  Pharmasan Labs, Inc..  Een selectieve en gevoelige vloeistofchromatografie-tandem-massaspectrometrie (LC-MS / MS) methode gekoppeld aan een efficiënte vaste fase-extractie op een microplaat van 96-putjes met gemengde modus kation-uitwisseling (MCX) 96-putjes werd ontwikkeld voor het meten van vrije 3-nitrotyrosine ( 3-NT) in menselijke urine met hoge doorvoer, die geschikt is voor klinische toepassingen. 

Video: Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

Cancer Research

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

Medicine

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

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 &#60; 2.5 &#181;m 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 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

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 ...

Environment

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Biochemistry

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Bioengineering

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

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