This work was in part supported by grants from the Singapore Ministry of Education (Tier 2: Grant ARC9/15), National Medical Research Council of Singapore (NMRC-OF-IRG-0003-2016), and NTU-NHG Ageing Research Grant (Grant ARG/14017). We would like to express our gratitude and most sincere thanks to Dr. Andrew Alpert and PolyLC team for kindly provided us with the packing materials that made possible this study.

1. Packing the Long-length Anion-exchange (LAX) Capillary Column
(Note: Although the LAX column can be in-home packed as we describe in this protocol, LAX columns are also commercially available, see Table of Materials and Reagents for further details).
<ol>
	<li>Suspend 50 mg of weak anion exchange packing material in 3.5 mL of packing buffer (Table 1) to prepare the slurry.</li>
	<li>Assemble the end of the capillary column (50 cm length - 200 &#181;m internal diameter (ID) tubing) using a female-to-female fitting, a ferule and a female nut. Place a screen 1/16&#34; OD of 1 micron pore size inside the female-to-female fitting. (Note: The screen used here must have smaller pore size than the particle size of the packing material to prevent leakage of the material from the column).</li>
	<li>Pack the capillary with the slurry using a pressure pump operated at 4,500 psi. (Note: Pack the column until the packing material is visible at the entry of the column).</li>
	<li>Assemble the other end of the capillary column as described in point 1.2.</li>
</ol>
2. Sample Preparation
This protocol outlines the application of LERLIC-MS/MS to analyze human brain tissues as model proteome. (Note: In case to use other tissues or proteomic samples, the sample preparation procedures should be adapted.)
<ol>
	<li>Tissue homogenization: 
	a. Wash brain tissues (50 to 100 mg) with 1x phosphate buffer solution for five minutes thrice. 
	b. Homogenize the tissue at 1:1:2.5 tissue/metallic beads/SDC homogenization buffer (Table 2) ratio (w/w/v) for 5 min at 4 &#176;C in safe-lock tubes at maximum intensity using a tissue homogenizer. (Note: SDC homogenization buffer may include protease inhibitors as previously indicated in3) 
	c. Centrifuge the tissue homogenate, obtained from the previous step, at 10,000 &#215; g, 4 &#176;C for 10 min and collect the supernatant into a new 1.5 mL tube. 
	d. Repeat steps b-c for the remaining pellets and combine the supernatants as many times as necessary until no pellet is observed. (Note: If you have to repeat the steps b-c more than two times, move the sample and the beads to a new safe-lock tube to prevent the loss of sample during the centrifugation step). 
	e. Quantify the protein concentration of the obtained homogenates by Bicinchoninic Acid assay (BCA)12.</li>
	<li>Sodium deoxycholate (SDC)-assisted in-solution tryptic digestion13 of brain homogenates. 
	a. Add a final concentration of 10 mM dithiothreitol (DTT) (using the stock solution indicated in Solution 5 of Table 3) to the obtained homogenate to initiate the reduction of protein disulfide bonds. 
	b. Incubate the homogenate during 30 min in a bath pre-set at 60 &#176;C. 
	c. Add to the homogenate a final concentration of 20 mM iodoacetamide (IAA) using the stock solution indicated in Solution 7 of Table 4. 
	d. Incubate the homogenate containing IAA at room temperature in the dark during 45 min. 
	e. Dilute the brain homogenate two-folds using dilution buffer (Solution 6 of Table 3). 
	f. Incubate the sample for a second reduction step during 30 min at 37 &#176;C. 
	g. Add sequencing-grade-modified trypsin dissolved in Solution 2 of Table 2 at protein-to-enzyme ratio 1:50 (w/w). 
	h. Digest the homogenate with trypsin by overnight incubation at 30 &#176;C. 
	i. Quench the enzymatic digestion on the next day by adding 0.5% final concentration of formic acid (FA). (Note: Addition of FA will cause precipitation of the SDC salts under acidic conditions.) 
	j. Gently vortex the samples containing SDC precipitates. 
	k. Pellet down the SDC salts by centrifuging the samples at 12,000 &#215; g, 4 &#176;C during 10 min. 
	l. Collect the supernatant and transfer the liquid to a clean new tube. (Note: Take care during the pipetting of this step to do not re-suspend and/or collect salts from the SDC pellet.) 
	m. Re-dissolve the SDC pellet in SDC re-dissolving buffer (Table 5) under vigorous vortexing for 1 min. 
	n. Repeat steps j-l twice to recover precipitated peptides and combine the supernatants. (Note: See Serra et al. 201613 for further details on the SDC-assisted in-solution tryptic digestion protocol adapted here.)</li>
	<li>Desalting of tryptic digested samples. 
	a. Perform desalting of digested samples using a 1g C-18 cartridge. (Note: The use of a big volume cartridge (5 mL) independently of the amount of protein obtained guarantees proper cleaning of remaining SDC salts in the samples prior to LC-MS/MS injection.) 
	b. Perform conditioning of the 1g C-18 cartridge with 5 mL of acetonitrile (ACN). 
	c. Pass 5 mL of clean-up buffer (Table 6) by the conditioned 1g C-18 cartridge to remove any remaining organic solvent. 
	d. Load the sample to the 1g C-18 cartridge. 
	e. Perform 3 - 5 clean-up steps to the sample in the 1g C-18 column using 5 mL of clean-up buffer each step. 
	f. Elute the desalted peptides from the 1g C-18 cartridge using 5 mL of elution buffer (Table 7). 
	g. Dry the eluted peptides in a vacuum concentrator. 
	c. Reconstitute the dried sample in a final volume of 200 &#181;L of elution buffer. (Note: Use vigorous and long (&#62; 10 min) vortexing followed by subsequent sonication in a sonication bath during 30 min to completely re-suspend the dried peptides in injection buffer.) 
	d. Adjust the injection volume of the sample for LERLIC-MS/MS to analyze between 1 to 3 &#181;g of protein.</li>
</ol>
3. One-dimension LERLIC-MS/MS Separation
<ol>
	<li>Liquid chromatography conditions: 
	Mobile phases: 
	A: 0.1% FA in water (Table 8). 
	B: 0.1% FA in ACN (Table 9) 
	Flow rate: 0.4 &#181;L/min 
	Column: LAX capillary column (50 cm length - 200 &#181;m ID) 
	a. Use the following 1200 min-gradient: 95% B for 40 min, 95 &#8722; 85% B for 434 min, 85 &#8722; 70% B for 522 min, 70 &#8722; 35% B for 124 min, 35 &#8722; 3% B for 45 min, isocratic at 3% B for 5 min, 3 &#8211; 95% B over 7 min and kept isocratic at 95% B for 23 min. 
	b. Perform separation of peptides using the LAX capillary as indicated in Serra &#38; Gallart-Palau et al.3 using ultra-high pressure liquid chromatography.</li>
	<li>Mass spectrometer configuration: 
	a. Configure the scan event details to perform data acquisition alternating between full Fourier transform-mass spectrometry (FT-MS) and Fourier transform-tandem mass spectrometry (FT-MS/MS) with the following parameters: 
	a.1. Data acquisition mode: positive 
	a.2 FT-MS parameters: 
	Mass range: 350 &#8722; 2,000 m/z 
	Resolution: 60,000 
	Microscans: 1 per spectrum 
	Automatic gain control: 1 &#215; 106 
	a.3 FT-MS/MS parameters: 
	Fragmentation mode: High-energy collisional dissociation (HCD) 
	a.4 Mass range: 150 &#8722; 2,000 m/z 
	a.5 Resolution: 30,000 
	a.6 Top N ions: 10 
	Microscans: 1 per spectrum 
	Charge state: &#62; 2+ 
	Isolation width: 2 Da 
	a.7 Automatic gain control: 1 &#215; 106 
	b. Perform mass spectrometry detection of peptides as indicated in3 using an orbitrap mass spectrometer equipped with a nanoelectrospray ion source working at 1.5 kV.</li>
</ol>
4. Data Analysis
<ol>
	<li>Perform protein database search to the LERLIC-MS/MS obtained data using the following parameters using a specific proteomic software: (Note: See3 for further details.) 
	a. Ion tolerance: 10 ppm 
	b. Fragment ion tolerance: 0.05 Da 
	c. False Discovery Rate (FDR): 1% 
	d. Database: UniProt Human database 
	e. Fixed modifications: Carbamydomethylation at Cys 
	f. Variable modifications (if required by the software): Oxidation (Met), Deamidation (Asn and Gln).</li>
	<li>Confident characterization and quantification of isomeric deamidated products in LERLIC-MS/MS data: 
	a. Export database search results from LERLIC-MS/MS to an spreedsheet software for analysis. 
	b. Find and extract the whole list of deamidated peptides and their non-deamidated counterparts. 
	c. Using the list of the obtained peptides as reference, extract the ion chromatograms (XICs) of these peptides using an appropiate software at 5 ppm of mass tolerance. (Note: See3 for further details on the software used for the extraction of XICs.) 
	d. Visually inspect the obtained XICs to identify the separated double-peak elution of the isomeric products as indicated3. (Note: Isomeric products of those peptides identified at MS/MS level can be easily found guided by the presence of two different retention times in the database search result.) 
	e. Relative quantification of the isomeric products from each deamidated site/peptide has to be performed based on the peak area of each identified isomer in the obtained XICs.</li>
</ol>

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The authors have nothing to disclose.

In this video-article we present a step-by-step protocol of LERLIC-MS/MS3, a method to perform in-depth characterization and to accurately determine the extent of protein deamidation and the enzymatic processes involved on this protein modification. LERLIC-MS/MS is based on the use of a long-length (50 cm) LAX under the principle of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)27. The use of a long-length column, as shown in our study<sup class="xre...

Deamidation is a protein posttranslational modification (PTM) that introduces a negative charge to the protein backbone through modification of asparagine (Asn) and/or glutamine (Gln) residues1. This modification while affecting Asn residues generates the isomeric products isoaspartic acid (isoAsp) and n-aspartic acid (Asp) at a common 3:1 ratio2. Notwithstanding, this ratio can be altered by the intervention of the repairing enzyme L-isoaspartyl methyltransferase (PIMT)3,4. Similarly, deamidation of Gln residues generates the isomeric gamma-glutamic acid (&#947;-Glu) and alpha-glutamic acid isoforms (&#945;-Glu) at an expected 1:7 ratio3,5, but this ratio can be shifted by the action of the ubiquitous enzyme transglutaminase 2 and other transglutaminases, including transglutaminase 1, an enzyme recently identified as associated with extracellular vesicles in the brain6.
The origin of deamidation can be either spontaneous or enzymatic, the former&#160;is especially common on Gln residues in which transglutaminases and other enzymes mediate&#160;inter/intra-molecular crosslinking via transamidation (see 3 for further details on Gln transamidation and its implications in several chronic and fatal human diseases). Therefore, deamidation is a PTM that has a crucial repercussion on the structure and function of affected molecules4,7,8 and requires an in-depth chemical characterization3 in the light of its diverse biochemical consequences including its service as molecular clock of aging9.
Although deamidation of Asn residues has been relatively well-characterized by bottom-up shotgun proteomics1,10, deamidation of Gln residues still does not have a suitable characterization method beyond the challenging analysis of model compounds by electron-based radical fragmentation11. We have recently developed a novel one-dimension shotgun proteomics strategy (LERLIC-MS/MS)3 that enables separation of Gln and Asn deamidation isoforms from complex biological samples and model compounds in a single analysis. LERLIC-MS/MS is based on the separation of tryptic digested peptides using a long-length (50 cm) ion exchange column (LAX) working on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode and coupled to tandem mass spectrometry (LC-MS/MS). This new analytical strategy has been used to characterize and relatively quantitate the extent of each deamidated residue in human brain tissues3. Nevertheless, the protocol outlined here will provide video imaging of LERLIC-MS/MS aimed to study the peculiarities of protein deamidation in the biochemical context of interest.
ETHICS STATEMENT
All procedures of this protocol have been approved by the institutional review board of the Nanyang Technological University in Singapore and have been performed in accordance to the institutional guidelines.

<table><tbody><tr><td>PolyCAT 3 &amp;micro;m 100-&amp;Aring; (bulk material)</td><td>PolyLC Inc.</td><td>Special order</td><td></td></tr><tr><td>Long-length ion exchange capillary column 50 cm - 200 &amp;micro;m ID</td><td>PolyLC Inc.</td><td>Special order</td><td></td></tr><tr><td>PEEKsil Tubing 1/16&quot; OD x 200 &amp;micro;m ID x 50 cm length</td><td>SGE Analytical Science under Trajan Scientific Australia&amp;nbsp;</td><td>620050</td><td></td></tr><tr><td>Female-to-female fitting for 1/16&quot; OD tubbing</td><td>Upchurch Scientific</td><td>UPCHF-125</td><td></td></tr><tr><td>Female nut for microferule</td><td>Upchurch Scientific</td><td>UPCHP-416</td><td></td></tr><tr><td>Microferule</td><td>Upchurch Scientific</td><td>UPCHF-132</td><td></td></tr><tr><td>Pressure Bomb NanoBaume</td><td>Western Fluids Engineering</td><td>SP-400</td><td></td></tr><tr><td>Shimadzu Prominence UFLC system</td><td>Shimadzu</td><td>Prominence UFLC</td><td></td></tr><tr><td>Bullet Blender</td><td>Next Advance</td><td>BBX24</td><td></td></tr><tr><td>Safe-lock tubes</td><td>Eppendorf&amp;nbsp;</td><td>T9661-1000EA</td><td></td></tr><tr><td>Stainless steel beads. 0.9 &amp;ndash; 2.0 mm. 1 lb. Non-sterile.</td><td>Next Advance</td><td>SSB14B</td><td></td></tr><tr><td>Table-top centrifuge&amp;nbsp;</td><td>Hettich Zentrifugen</td><td>Rotina 380 R</td><td></td></tr><tr><td>Standard Digital Heated Circulating Bath, 120VAC</td><td>PolyScience 8006 6L</td><td>8006A11B</td><td></td></tr><tr><td>Sep-pack c18 desalting cartridge 50 mg</td><td>Waters</td><td>WAT020805</td><td></td></tr><tr><td>Vacumm concentrator</td><td>Eppendorf&amp;nbsp;</td><td>Concentrator Plus System</td><td></td></tr><tr><td>Dionex UltiMate 3000 UHPLC&amp;nbsp;</td><td>Dionex</td><td>UltiMate 3000 UHPLC&amp;nbsp;</td><td></td></tr><tr><td>Orbitrap Elite mass spectrometer</td><td>Thermo Fisher Scientific Inc.</td><td>ORBITRAP ELITE</td><td></td></tr><tr><td>Michrom Thermo CaptiveSpray&amp;nbsp;</td><td>Michrom-Bruker Inc.</td><td>TCSI-SS2</td><td></td></tr><tr><td>Incubator INCUCELL&amp;nbsp;</td><td>MMM Group</td><td>INCUCELL111</td><td></td></tr><tr><td>Sequencing-grade modified trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Protease inhibitor cocktail tablets</td><td>Roche</td><td>11836170001 (ROCHE)</td><td></td></tr><tr><td>Phosphate buffer solution 10x (diluted to 1x)</td><td>Sigma-Aldrich</td><td>P5493</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A1542</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>i6125</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507 (HONEYWELL)</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Sigma-Aldrich</td><td>338818 (HONEYWELL)</td><td></td></tr><tr><td>Acetonitrile HPLC grade</td><td>Sigma-Aldrich</td><td>675415</td><td></td></tr><tr><td>Isopropanol HPLC grade</td><td>Sigma-Aldrich</td><td>675431</td><td></td></tr><tr><td>Water HPLC grade</td><td>Sigma-Aldrich</td><td>14263</td><td></td></tr></tbody></table>

lerlic-ms/ms for in-depth characterization and quantification of glutamine and asparagine deamidation in shotgun proteomics

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in human pathology and other biochemical contexts. In order to perform characterization of protein deamidation, we have recently developed a novel long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method which can separate the glutamine (Gln) and asparagine (Asn) isoform products of deamidation from model compounds to highly complex biological samples. LERLIC-MS/MS is, therefore, the first shotgun proteomics strategy for the separation and quantification of Gln deamidation isoforms. We also demonstrate, as a novelty, that the sample processing protocol outlined here stabilizes the succinimide intermediate allowing its characterization by LERLIC-MS/MS. Application of LERLIC-MS/MS as shown in this video article can help to elucidate the currently unknown molecular arrays of protein deamidation. Additionally, LERLIC-MS/MS provides further understanding of the enzymatic reactions that encompass deamidation in distinct biological backgrounds.

Deamidation of Gln and Asn residues is considered a degenerative protein modification (DPM) implicated in several chronic and fatal diseases14. It has been demonstrated that this PTM can predict the half-life and degradative states of antibodies and other molecules in the human body and similar biological backgrounds1,15. The significance of protein deamidation, in fact, goes beyond the biomedical context, ...

Watch this Scientific Journal Video about LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics at JoVE.com

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Here we present a step-by-step protocol of the long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method. This is a novel methodology that enables for the first time quantification and characterization of the glutamine and asparagine deamidation isoforms by shotgun proteomics.

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

lerlic-msms-for-depth-characterization-quantification-glutamine

Research

JoVE Journal

Biochemistry

8.1K Views.  Nanyang Technological University.  Hier geven we een stap-voor-stap protocol van de lange lengte elektrostatische repulsie-hilic-tandem massaspectrometrie (LERLIC-MS / MS) methode. Dit is een nieuwe methode die het mogelijk maakt voor het eerst kwantificering en karakterisering van glutamine en asparagine deamidering isovormen door shotgun proteomics. 

Video: LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Chemistry

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 ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, which can take up significant person-hours of bench labor and present a source of batch-to-batch variability. Lab automation with pipetting robots can reduce manual work, maximize throughput, and increase research reproducibility. Still, the steep starting prices of standard automation stations make them unaffordable for many academic laboratories. This article describes a proteomics sample preparation workflow using an affordable, open-source automation system (The Opentrons OT-2), including instructions for setting up semi-automated protein reduction, alkylation, digestion, and clean-up steps; as well as accompanying open-source Python scripts to program the OT-2 system through its application programming interface.

Detailed protocol and three Python scripts are provided for operating an open-source robotic liquid handling system to perform semi-automated protein sample preparation for mass spectrometry experiments, covering detergent removal, protein digestion, and peptide desalting steps.

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, ...

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson&#39;s disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.

A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

Neuroscience

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Biology

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

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