Name: improved in-gel reductive &#946;-elimination for comprehensive o-linked and sulfo-glycomics by mass spectrometry
Uploaded: 2014-11-20T13:00:00
Description: Watch this Scientific Journal Video about Improved In-gel Reductive Î²-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry at JoVE.com

This work was supported by the grant P01HL107151 from the NHLBI/NIH. The authors also gratefully acknowledge the support and access to instrumentation provided through grant P41GM103490 from the NIGMS/NIH.

NOTE: Lab Safety Concerns
In keeping with standard laboratory best practices, observe the following. Store all organic solvents in appropriate locations. Keep all waste materials in chemical waste containers with clear labeling of chemical compositions. As several reagents used in these protocols are potential carcinogens or generate volatile combustible gases, handle all reagents in a fume hood with ventilation. Wear personal protective equipment such as gloves, lab coat, and...

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Combining in-gel reductive &#946;-elimination with aqueous-organic extraction enhances the sensitivity and depth of structural data that can be acquired for characterizing sulfated and non-sulfated O-linked glycans harvested from small amounts of mucin-type glycoproteins resolved from other proteins by SDS-PAGE. The essential advances of the technical approaches presented in this study are: (a) facile removal of gel derived contaminants by simple washing steps; (b) quantitative recovery of permethylated sulfoglycans in t...

Glycosylation is an essential protein post-translational modification, contributing to organismal physiology, tissue pathology, and cellular recognition 1-3. Despite major advances in analytical glycoscience, characterizing the complete diversity of glycans on a specific protein remains an extremely challenging task, especially on proteins isolated from primary biological sources. Nonetheless, the microheterogeneity of glycoprotein glycans frequently affects functional interactions with other proteins. Therefore, characterization of glycan diversity is essential for understanding the physiological significance of cellular and tissue glycosylation 4,5. In order to understand the contribution of glycoprotein glycosylation to tissue physiology and pathophysiology, robust, sensitive, and comprehensive glycomic analytical techniques have become increasingly important. In proteomic analysis, protein identifications are generally achieved by LC-MS/MS analysis of tryptic peptides 6. Protein digestion can be carried out using a purified protein or proteins resolved by SDS-PAGE following in-gel digestion with proteases such as trypsin 7-9. Pre-enrichment of the protein mixture by SDS-PAGE enhances the depth and accuracy of protein ID. The development of analogous strategies for glycomic analysis of glycoprotein glycosylation lies at the forefront of glycoscience.
The two major classes of glycans are attached to protein backbones through either N-linkage or O-linkage. N-linked glycans are attached to asparagine (Asn) residues found as part of a sequon defined as Asn-X-Ser/Thr/Cys (X is any amino acid except proline), and can be released by enzymatic digestion with peptide-N-glycanase (PNGaseF or A), either in solution, in-gel, or on-blot 10-12. O-linked glycans are mainly attached to serine (Ser) or threonine (Thr) residues. However, only one enzyme has been identified that is capable of releasing O-linked glycans from glycoprotein and it has an extremely limited glycan specificity, releasing only the simplest O-linked glycans. Chemical release strategies remain the method of choice for comprehensive release of O-linked glycans from glycoproteins. Reductive or non-reductive &#946;-elimination, or hydrazinolysis are well-characterized chemical release techniques and are currently the most commonly used approaches for releasing O-linked glycans from glycoproteins 13,14. Although reductive &#946;-elimination has been used to release O-linked glycans from glycoproteins separated by SDS-PAGE, previous approaches required HPLC separation for subsequent analysis 15-17.
Multidimensional MS (MSn) analysis currently provides the richest source of structural data for characterizing glycans released from glycoproteins isolated in the amounts expected from most biological sources. The depth of MS-based structural characterization is greatly facilitated by permethylating the released glycans prior to their analysis. Permethylation enhances ionization and tends to equalize molar signal responses across a broad range of glycan structures 18,19. In addition, permethylation unambiguously tags terminal and substituted monosaccharide moieties with distinctive masses, thereby enhancing structural elucidation 20-23. For example, acidic glycans are generally difficult to detect as non-permethylated species by MS. Although acidic glycans can be detected in negative ion mode by MS, it is impossible to detect both acidic and neutral glycans in the same ion mode. A major advantage of glycan permethylation is that all of the free hydroxyl groups (OH) on a glycan&#8217;s monosaccharide substituents will be capped with a methyl group (OCH3 or OMe), thus a sialylated glycan&#8217;s charges are neutralized, making them as detectable as permethylated neutral (asialo) glycans. However, the hydroxyls of sulfate moieties on sulfoglycans are resistant to permethylation, resulting in retention of anionic charge, which suppresses ionization and decreases sensitivity. This suppression currently prevents comprehensive glycomic analysis of very complex glycoproteins such as mucins, which carry a high abundance of sulfated glycans 24-26.
Recent reports on purifying sulfated glycans used charged, reverse-phase chromatography to purify and separate permethylated glycans prior to MALDI analysis. This method relies on complete separation of sulfated and non-sulfated glycans using different mobile phases for elution, which we have found to be less stringent than organic phase partitioning. Therefore, new techniques suitable for the detection and enrichment of sulfoglycans are presented here. These techniques allow for the quantitative recovery of sulfated glycans in the aqueous phase following water:DCM (dichloromethane) extraction, which is routinely performed at the end of glycan permethylation reactions 27. Importantly, this robust separation of permethylated sulfoglycans from a mixture of permethylated non-sulfated glycans concomitantly enriches for charged species while also simplifying MS2 fragmentation patterns. A comprehensive protocol for improved in-gel O-linked glycan analysis is also presented. The improved protocol enhances glycan recovery, increases the structural information obtainable through MSn analysis of permethylated glycans, and improves the sensitivity of sulfoglycomic analyses applied to essential glycoproteins isolated from biological sources.
This protocol is intended for O-linked glycan analysis of whole glycoprotein extracts or of a specific glycoprotein of interest resolved by SDS-PAGE and is composed of three experimental procedures; A) gel clean-up, B) in-gel reductive &#946;-elimination, and C) glycan permethylation. The goal is to obtain comprehensive O-linked glycomic data for glycoproteins harvested from primary sources of biological interest (Figure 1). Glycoproteins separated by SDS-PAGE are visualized by staining and bands of interest are excised and the resulting gel band is sliced into small pieces. The gel pieces are destained and subjected to ethyl acetate washes to remove gel contaminants (Figure 2A). Glycan release is achieved by in-gel reductive &#946;-elimination (Figure 2B) and the released glycans are permethylated. Aqueous-organic extraction following permethylation quantitatively partitions the anionic sulfated glycans away from non-sulfated neutral glycans (Figure 2C). In-gel reductive &#946;-elimination coupled to aqueous-organic extraction enables the characterization of O-linked glycans and sulfoglycans released from small amounts of glycoprotein separated by SDS-PAGE. The strategic overview is summarized in Figure 1 and the details are shown in Figure 2. In addition, a portion of the destained and washed gel pieces can be used for protein ID by standard LC-MS/MS proteomic techniques.

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			<td height="21" style="height:21px;width:231px;">Name of Material/ Equipment</td>
			<td style="width:155px;">Company</td>
			<td style="width:124px;">Catalog Number</td>
			<td style="width:303px;">Comments/Description</td>
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			<td height="19" style="height:19px;width:231px;">Water, deionized water</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>270733-4L</td>
			<td style="width:303px;">CHROMASOLV, for HPLC</td>
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			<td height="21" style="height:21px;width:231px;">Acetic acid, Glacial</td>
			<td style="width:155px;">Fisher</td>
			<td>A38-500</td>
			<td style="width:303px;">Certified ACS&#8805;99.7 w/w %</td>
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			<td height="21" style="height:21px;width:231px;">Methanol</td>
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			<td>34860-4L-R</td>
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			<td height="21" style="height:21px;width:231px;">Ammonium bicarbonate</td>
			<td style="width:155px;">Fluka</td>
			<td>09830-500G</td>
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			<td>34998-4L</td>
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			<td height="21" style="height:21px;width:231px;">Ethyl acetate</td>
			<td style="width:155px;">Fluka</td>
			<td>34972-1L-R</td>
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			<td height="21" style="height:21px;width:231px;">Sodium borohydride</td>
			<td style="width:155px;">Aldrich</td>
			<td>213462-25G</td>
			<td style="width:303px;">ReagentPlus, 99%, Step 2</td>
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			<td height="42" style="height:42px;width:231px;">Iodomethane</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>289566-100G</td>
			<td style="width:303px;">ReagentPlus, 99.5%,&#160; Step 6.&#160; Store at 4 &#176;C until use.&#160; Sit at room temperature before use.</td>
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			<td height="42" style="height:42px;width:231px;">Sodium hydroxide solution, 50% w/w</td>
			<td style="width:155px;">Fisher</td>
			<td>SS254-1</td>
			<td style="width:303px;">Certified, Step 6</td>
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			<td height="21" style="height:21px;width:231px;">Dimethyl sulfoxide, anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>276855-1L</td>
			<td style="width:303px;">Anhydrous, &#8805;99.9%, Step 6</td>
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			<td height="21" style="height:21px;width:231px;">Methanol, anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>322415-100ML</td>
			<td style="width:303px;">Anhydrous, 99.8%, Step 6</td>
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			<td height="42" style="height:42px;width:231px;">Dichloromethane</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>34856-4L</td>
			<td style="width:303px;">CHROMASOL&#174;, for HPLC, &#8805;99.8%, contains amylene as stabilizer, Step 7</td>
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			<td height="25" style="height:25px;width:231px;">Dowex 50WX8 hydrogen formhydrogen form 100-200&#160;mesh</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td>217506-500G</td>
			<td style="width:303px;">Step 3</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:231px;">AG 50W-X8 Resin&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td>142-1441</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:231px;">BAKERBOND spe 1 ml x 100 mg Solid Phase Extraction Column, PP, Octadecyl (C18) Reverse Phase</td>
			<td style="width:155px;">JT Baker</td>
			<td>JT-7020-01</td>
			<td style="width:303px;">Step 5 and 8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">7.5% Mini-PROTEAN TGX Precast Gel&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td></td>
			<td style="width:303px;">Step 1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Bio-Safe Coomassie Stain&#160;</td>
			<td style="width:155px;">Bio-Rad</td>
			<td>161-0786</td>
			<td style="width:303px;">G-250, Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Silver Stain Kit for Mass Spectrometry</td>
			<td style="width:155px;">Pierce</td>
			<td>24600</td>
			<td style="width:303px;">Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Oligosaccharides Kit (Maltotriose, Dp3: R474140)</td>
			<td style="width:155px;">Supelco&#160;</td>
			<td>47265</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Oligosaccharides Kit (Maltotetraose, Dp4: R474135)</td>
			<td style="width:155px;">Supelco&#160;</td>
			<td>47265</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Disposable Pasteur Pipets, Glass, Short Tip</td>
			<td style="width:155px;">VWR</td>
			<td>14673-010</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">PYREX 13 x 100 mm Disposable Round Bottom Threaded Culture Tubes</td>
			<td style="width:155px;">Corning</td>
			<td>99447-13</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">PYREX 16 x 125 mm Disposable Round Bottom Threaded Culture Tubes</td>
			<td style="width:155px;">Corning</td>
			<td>99447-16</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Phenolic Caps/Closures with PTFE-Faced Rubber Liner</td>
			<td style="width:155px;">Kimble</td>
			<td>45066C-13</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Phenolic Caps/Closures with PTFE-Faced Rubber Liner</td>
			<td style="width:155px;">Kimble</td>
			<td>45066C-15</td>
			<td style="width:303px;">Wash before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81265</td>
			<td style="width:303px;">volume 500&#160;&#956;L, needle size 22 ga&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81165</td>
			<td style="width:303px;">volume 250&#160;&#956;L, needle size 22 ga</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>81065</td>
			<td style="width:303px;">volume 100&#160;&#956;L, needle size 22s ga&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton HPLC syringe</td>
			<td style="width:155px;">HAMILTON</td>
			<td>80965</td>
			<td style="width:303px;">volume 50&#160;&#956;L, needle size 22s ga</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hamilton Calibrated Syringes</td>
			<td style="width:155px;">HAMILTON</td>
			<td>80300</td>
			<td style="width:303px;">volume 10&#160;&#956;L, needle size 26s ga&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">Petri Dish Glass 100mm x 15mm</td>
			<td style="width:155px;">GSC INTERNATIONAL INC</td>
			<td>1500-4</td>
			<td style="width:303px;">Wash before use, Step 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Bard-Parker Surgical Blades</td>
			<td style="width:155px;">Fisher</td>
			<td>371310</td>
			<td style="width:303px;">Step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Reacti-Therm Heating/Stirring Module</td>
			<td style="width:155px;">Pierce</td>
			<td>18870</td>
			<td style="width:303px;">Step 1, 4 and 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Heating Blocks</td>
			<td style="width:155px;">Fisher</td>
			<td>125D</td>
			<td style="width:303px;">Step 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Pyrex fiber glass wool borosilicate pore size 8&#160;&#956;m</td>
			<td style="width:155px;">Aldrich</td>
			<td>CLS3950</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fused Silica CutterTubing cutter</td>
			<td style="width:155px;">alltech</td>
			<td>3194</td>
			<td style="width:303px;">Step 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Multi-tube vortexers&#160;</td>
			<td style="width:155px;">VWR</td>
			<td>444-7063</td>
			<td style="width:303px;">Step 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Lyophilizer 25EL Freezemobile</td>
			<td style="width:155px;">Virtis</td>
			<td>25EL</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Centrifuge</td>
			<td style="width:155px;">VWR</td>
			<td>Clinical 50</td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;"></td>
			<td style="width:155px;"></td>
			<td></td>
			<td style="width:303px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">LTQ Orbitrap Discovery</td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td></td>
			<td style="width:303px;">0</td>
		</tr>
	</tbody>
</table>

improved in-gel reductive &#946;-elimination for comprehensive o-linked and sulfo-glycomics by mass spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Effect of Ethyl Acetate Treatment Prior to In-gel Reductive &#946;-Elimination
A representative mass spectrum of permethylated O-linked glycan samples released from bovine mucin using in-gel reductive &#946;-elimination is shown in Figure 3. The EtOAc wash of the gel pieces effectively removes SDS and polyacryl contaminants which interfere with subsequent MS analysis 27.
<img alt="Fig...

Watch this Scientific Journal Video about Improved In-gel Reductive Î²-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry at JoVE.com

Improved In-gel Reductive &amp;#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

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Unit 1

Nucleophilic Substitution and Elimination Reactions of Alkyl Halides

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Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

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 ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Chromatographic Fingerprinting by Template Matching for Data Collected by Comprehensive Two-Dimensional Gas Chromatography

Data processing and evaluation are critical steps of comprehensive two-dimensional gas chromatography (GCxGC), particularly when coupled to mass spectrometry. The rich information encrypted in the data may be highly valuable but difficult to access efficiently. Data density and complexity can lead to long elaboration times and require laborious, analyst-dependent procedures. Effective yet accessible data processing tools, therefore, are key to enabling the spread and acceptance of this advanced multidimensional technique in laboratories for daily use. The data analysis protocol presented in this work uses chromatographic fingerprinting and template matching to achieve the goal of highly automated deconstruction of complex two-dimensional chromatograms into individual chemical features for advanced recognition of informative patterns within individual chromatograms and across sets of chromatograms. The protocol delivers high consistency and reliability with little intervention. At the same time, analyst supervision is possible in a variety of settings and constraint functions that can be customized to provide flexibility and capacity to adapt to different needs and goals. Template matching is shown here to be a powerful approach to explore extra-virgin olive oil volatilome. Cross-alignment of peaks is performed not only for known targets, but also for untargeted compounds, which significantly increases the characterization power for a wide range of applications. Examples are presented to evidence the performance for the classification and comparison of chromatographic patterns from sample sets analyzed under similar conditions.

This protocol presents an approach to fingerprint and explore multi-dimensional data collected by comprehensive two-dimensional gas chromatography coupled to mass spectrometry. Dedicated pattern recognition algorithms (template matching) are applied to explore the chemical information encrypted in the extra-virgin olive oil volatile fraction (i.e., volatilome).

Data processing and evaluation are critical steps of comprehensive two-dimensional gas chromatography (GCxGC), particularly when coupled to mass ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;s structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...