We are grateful to the individuals with BTHS and their families for participating in our research. We thank the Barth Syndrome Foundation US and the Barth Syndrome UK Trust for their support and for helping with the collection of the blood samples at the annual meeting in Bristol. This study was funded by Barth Syndrome Foundation US, Barth Italia Onlus, and Apulia Region.

Blood samples of healthy donors and heart failure patients were collected at the Policlinic Hospital of Bari (Italy), while samples of BTHS patients were obtained by the National Health Service UK BTHS clinic at Bristol Royal Hospital for Children (UK). Written informed consent of healthy donors, patients, and parents (where appropriate) and approvals by the respective ethics committees were obtained.
NOTE: If not used immediately, blood (in K-EDTA gel tube) can be stored at 4 &#176;C for up to 24-48 h.
1. Isolation of leukocytes by dextran sedimentation of red blood cells (RBCs)
<ol>
	<li>Put blood samples (in K-EDTA) on an orbital shaker for 10 min to mix blood.</li>
	<li>To 0.9 mL of whole blood in a 1.5 mL tube, add 0.1 mL of 20% dextran solution (molecular mass &#62; 100 kDa, in 0.9% NaCl). Pipette and disperse the suspension gently 20 times while avoiding air bubbles that would otherwise retain RBCs at the top of the tube. Let RBCs sediment for about 1 h at room temperature.</li>
	<li>Using a syringe, collect and transfer the yellow supernatant to a 15 mL tube, and then centrifuge at 400 x g for 15 min at room temperature (no brake, swing-bucket rotor).</li>
	<li>Discard the supernatant and resuspend the pellet containing mainly leukocytes and residual RBCs in 0.6 mL of ice-cold bi-distilled water (ddH2O).</li>
	<li>After ~15 s, add 0.2 mL of 0.6 M KCl to the cell suspension to restore the correct osmolarity. The short osmotic shock will lyse RBC and leave leukocytes intact. Adjust the final volume to 2.5 mL with 1x PBS.</li>
	<li>Centrifuge the suspension at 400 x g for 15 min at room temperature (no brake, swing-bucket rotor). Discard the supernatant and wash the leukocyte pellet again with 2.5 mL of 1x PBS.</li>
	<li>Centrifuge as in step 1.6 and discard the supernatant. Resuspend the pellet containing leukocytes in 200 &#181;L of sterilized ddH2O. 
	NOTE: In the authors&#39; experience, this corresponds to a protein content ranging from 200 &#181;g to 400 &#181;g.</li>
	<li>Freeze the suspension at -80 &#176;C or directly perform lipid extraction as follows.</li>
</ol>
2. &#34;Mini-extraction&#34; of lipids from isolated leukocytes
<ol>
	<li>Transfer 20 &#181;L of leukocytes suspension (about 20-40 &#181;g proteins) to a 1.5 mL tube and spin at 16,000 x g for 30 s.</li>
	<li>Discard the supernatant, add 10 &#181;L of CHCl3 to the remaining pellet, and pipette repeatedly to promote lipid extraction.</li>
	<li>Finally, add 10 &#181;L of 9-aminoacridine (9-AA) matrix solution (10 mg/mL 9-AA in 2-propanol/acetonitrile, 60:40 v/v) to the pellet in CHCl3. Pipette and disperse repeatedly to mix.</li>
	<li>Spin the solution containing lipids in CHCl3 and 9-AA at 16,000 x g for 30 s, and then deposit the supernatant as droplets of 0.35 &#181;L (three replicates for each sample) on the MALDI target (sample plate) to be analyzed (&#39;dried droplet&#39; deposition method).</li>
	<li>Let the droplets air dry at least for 10-15 min.</li>
</ol>
3. Lipid analysis by MALDI-TOF/MS
<ol>
	<li>Acquire mass spectra of samples in triplicates on a MALDI-TOF mass spectrometer.</li>
	<li>After calibration with lipid standards (see Table of Materials), set the analyses in the negative ion mode and optimize the detection m/z range from 200 Th to 2,000 Th for small molecule analysis.</li>
	<li>Keep the laser fluence 5% above the threshold (of CL and MLCL) to have a S/N (at least 2).</li>
	<li>Acquire spectra in reflector mode using delayed pulsed extraction. For each mass spectrum, average 2,000 single laser shots (sum of 4 x 500). Apply gated matrix suppression to 400 Th to prevent detector saturation.</li>
</ol>
4. How to calculate the (MLCL + CLi)/CLm ratio
<ol>
	<li>Run the MALDI-TOF/MS instrument software (see Table of Materials) to analyze the acquired spectra.</li>
	<li>Using the Open command, open the Spectrum Browser dialog that allows selecting and loading the spectrum of interest.</li>
	<li>On the menu bar, click on the icons Smooth Mass Spectrum and Subtract Mass Spectrum Baseline.</li>
	<li>Click on File &#62; Export &#62; Mass Spectrum and choose the ASCII format for exporting the spectrum as a two-column table with pairs of data points: m/z (x) and intensity (y). Copy and paste the coordinates in a spreadsheet program (*.xls).</li>
	<li>Repeat steps 4.2 to 4.4 for the triplicates of each analyzed sample, pasting the coordinates in the same spreadsheet program file as shown in Figure 1 (Step 1).</li>
	<li>Following the x-ranges shown in Figure 1 (Step 2) for each species listed, calculate the sum of y1, y2, and y3 values (triplicates) by the SUM function to get the peak area (Figure 1; Step 3).</li>
	<li>Perform the average of the triplicates area values by AVERAGE function (Figure 1; Step 3).</li>
	<li>Place the average area values for each species in the column as shown in Figure 1 (Step 2).</li>
	<li>In order to consider only the first isotopologue of the CLm 72:7 species as in28, calculate the isotopic correction for the overlapping between the M + 2 isotopologue of the CLm 72:8 and the monoisotopic peak of CLm 72:7 as shown in Figure 1 (Step 4).</li>
	<li>Finally, calculate the (MLCL + CLi)/CLm ratio as shown in Figure 1 (Step 5).</li>
</ol>

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barth+syndrome.">Barth syndrome.</a> Orphanet Journal of Rare Diseases. 8, 23 (2013).</li><li>Zegallai, H. M., Hatch, G. 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All authors declare that the study was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

Barth syndrome is an inborn error of metabolism and a life-changing condition that is likely to be under-diagnosed2,6. As mentioned before, a contributing factor may be the lack of a straightforward diagnostic test. Here, a simple and fast method to measure MLCL/CL ratio by MALDI-TOF/MS in leukocytes for BTHS screening was described. Moreover, MALDI-TOF mass spectrometers are widely distributed among clinical laboratories worldwide and do not require high an...

Barth syndrome (BTHS) is a rare X-linked disease characterized by early-onset cardiomyopathy, skeletal muscle myopathy, growth delay, neutropenia, variable mitochondrial respiratory chain dysfunction, and abnormal mitochondrial structure1,2,3,4,5. BTHS has a prevalence of one case per million males with currently less than 250 known cases worldwide, though it is widely accepted that the disease is underdiagnosed2,6. BTHS results from loss-of-function mutations of the Tafazzin (TAFAZZIN) gene localized to chromosome Xq28.127,8 causing deficient remodeling of the mitochondrial phospholipid cardiolipin (CL), a process that normally leads to a highly symmetric and unsaturated acyl composition9,10. CL has been considered the signature lipid of mitochondria, where it is an important constituent of the inner membrane, vital for oxidative phosphorylation (i.e., mitochondrial energy metabolism), supercomplex formation, protein import, and involved in mitochondrial dynamics, mitophagy, and apoptosis11,12,13,14,15,16. Upon TAFAZZIN loss-of-function, CL remodeling fails and specific phospholipid abnormalities arise in mitochondria of BTHS patients: mature CL level (CLm) is decreased, while increased levels of monolysocardiolipin (MLCL) and altered CL acyl composition (i.e., immature CL species, CLi) occur. This brings to a dramatic increase of the MLCL/CL ratio17.
Diagnosis of BTHS is often difficult, as the disorder presents extremely variable clinical and biochemical features and may differ between affected individuals from the same family and within a patient over time3,5. Many BTHS boys show a very high level of urinary excretion of 3-methylglutaconic acid (3-MGCA), but the urine level may be normal or only mildly increased in patients over time3. However, increased 3-MGCA is a feature of various other mitochondrial and non-mitochondrial disorders, such as 3-methylglutaconyl-CoA hydratase deficiency (AUH defect), 3-methylglutaconic aciduria, dystonia-deafness, encephalopathy, Leigh-like (MEGDEL) syndrome, Costeff syndrome, and dilated cardiomyopathy with ataxia (DCMA) syndrome18,19. Hence, the poor specificity of 3-MCGA as a marker for BTHS and the enormous variability in patients render the biochemical diagnosis ambiguous.
Moreover, over 120 different TAFAZZIN mutations have been described causing the disorder5 and, therefore, a genetic diagnosis can be complicated, slow, and expensive. Moreover, molecular analysis of the TAFAZZIN gene can lead to false-negative results in the presence of mutations in noncoding or regulating sequences3. BTHS can be unambiguously tested by determining the relative amounts and distribution of (monolyso-)CL species and confirmed by TAFAZZIN gene sequencing or vice versa.
A practical test for diagnosis is the measurement of the MLCL/CL ratio by High-Performance Liquid Chromatography (HPLC) and Electro Spray Ionization / Mass spectrometry (ESI/MS) analysis in blood spot20,21. Measuring CL level alone is not adequate for diagnosis as some patients have near-normal levels of CL but altered MLCL/CL ratio. Therefore, measurement of MLCL/CL ratio has 100% sensitivity and specificity for BTHS diagnosis21. Another validated method based on HPLC and ESI/MS analysis has been set up on leukocytes22, but the complex chromatographic techniques for separation of lipids previously extracted and the expensiveness of the instruments restrict this analysis to a few clinical laboratories. All these factors, together with the lack of a straightforward diagnostic test, have contributed to the under-diagnosis of the condition.
MALDI-TOF/MS is a further valid tool in lipid analysis23,24.This analytical technique can be used to directly obtain lipid profiles of various biological samples, thus skipping extraction and separation steps25,26,27,28,29, including in tissue sections for MS Imaging applications30. Given this advantage, a simple and fast method to diagnose BTHS by profiling mitochondrial CL in intact leukocytes with MALDI-TOF/MS was developed28. Leukocyte isolation from only 1 mL of whole blood by erythrocyte sedimentation and lysis is straightforward and does not require special equipment or reagents. Furthermore, a fast lipid &#34;mini-extraction&#34; protocol applicable to minute amounts of leukocytes was described to warrant the successful acquisition of spectra having cleaner MS signals with a higher signal-to-noise ratio (S/N) than in those obtained from intact leukocytes28. This further step takes little time and allows for analyses to be reproducible even when carried out on MS instruments with poor sensitivity. In summary, the analytical method described here requires minimal sample preparation because time-consuming and labor-intensive chromatographic lipid separation can be skipped, thereby speeding up the test.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,1&#8242;,2,2&#8242;-tetratetradecanoyl cardiolipin</td>
			<td>Avanti Polar Lipids</td>
			<td>750332</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,1&#8242;2,2&#8242;-tetra- (9Z-octadecenoyl) cardiolipin</td>
			<td>Avanti Polar Lipids</td>
			<td>710335</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,2-di- (9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>878130</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,2-ditetradecanoyl-sn-glycero-3-phosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>830845</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,2-ditetradecanoyl-snglycero-3-phospho-(1&#8242;-rac-glycerol)</td>
			<td>Avanti Polar Lipids</td>
			<td>840445</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,2-ditetradecanoyl-sn-glycero-3-phospho-L-serine</td>
			<td>Avanti Polar Lipids</td>
			<td>840033</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>2-Propanol, ACS reagent, &#8805;99.5%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>190764</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Aminoacridine hemihydrate, 98%</td>
			<td>Acros Organics</td>
			<td>134410010</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, ACS reagent, &#8805;99.5%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>360457</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform, ACS reagent, &#8805;99.8%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>319988</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran from Leuconostoc spp. Mr 450,000-650,000</td>
			<td>Merck Life Science S.r.l.</td>
			<td>31392</td>
			<td></td>
		</tr>
		<tr>
			<td>Flex Analysis 3.3</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>MALDI-TOF mass spectrometer Microflex LRF</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Office</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>OmniPur 10X PBS Liquid Concentrate</td>
			<td>Merck Life Science S.r.l.</td>
			<td>6505-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, ACS reagent, 99.0-100.5%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride, ACS reagent, &#8805;99.0%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>S9888</td>
			<td></td>
		</tr>
	</tbody>
</table>

fingerprinting cardiolipin in leukocytes by mass spectrometry for a rapid diagnosis of barth syndrome

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial role in the functioning of the inner membrane. Its metabolite monolysocardiolipin (MLCL) is physiologically nearly absent in the lipid extract of animal cells and its appearance is the hallmark of the Barth syndrome (BTHS), a rare and often misdiagnosed genetic disease that causes severe cardiomyopathy in infancy. The method described here generates a "cardiolipin fingerprint" and allows a simple assay of the relative levels of CL and MLCL species in cellular lipid profiles. In the case of leukocytes, only 1 mL of blood is required to measure the MLCL/CL ratio via matrix-assisted laser desorption ionization - time-of-flight/mass spectrometry (MALDI-TOF/MS) just within 2 h from blood withdrawal. The assay is straightforward and can be easily integrated into the routine work of a clinical biochemistry laboratory to screen for BTHS. The test shows 100% sensitivity and specificity for BTHS, making it a suitable diagnostic test.

In this study, a simple and rapid method for isolating leukocytes from 1 mL of whole blood and obtaining CL fingerprinting by MALDI-TOF/MS has been described (see Figure 2). Figure 3 shows the comparison of representative CL fingerprinting of leukocytes, obtained from control subjects and BTHS young boys, in the CL and MLCL mass (m/z) range. Table 1 lists CL and MLCL species detected in these mass spectra.
De...

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Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

This protocol shows how to obtain a mass spectrometric "fingerprint" of leukocyte cardiolipin for the diagnosis of Barth syndrome. The assessment of elevated monolysocardiolipin to cardiolipin ratio discriminates patients with Barth syndrome from control heart failure patients with 100% sensitivity and specificity.

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial ...

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Research

JoVE Journal

Biochemistry

2.2K Views.  Swansea University. This protocol shows how to obtain a mass spectrometric "fingerprint" of leukocyte cardiolipin for the diagnosis of Barth syndrome. The assessment of elevated monolysocardiolipin to cardiolipin ratio discriminates patients with Barth syndrome from control heart failure patients with 100% sensitivity and specificity.

Video: Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

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 ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...