We acknowledge MHRD-UAY Project (UCHHATAR AVISHKAR YOJANA), project #34_IITB to SS and MASSFIITB Facility at IIT Bombay supported by the Department of Biotechnology (BT/PR13114/INF/22/206/2015) to carry out all MS-related experiments.

This study was reviewed and approved by institutional review boards and the ethics committee of the Indian Institute of Technology Bombay (IITB-IEC/2016/026). The patients/participants provided their written consent to participate in this study.
1. Tissue lysate preparation
NOTE: Perform all the following steps on the ice to keep the proteases inactive. Make sure the scalpels and any tubes used are sterile to avoid any cross-contamination.
<ol>
	<li>Take ~30 mg of tissue in a bead beating tube, add 200 &#181;L of 1x phosphate buffer saline (PBS) and vortex it. 
	NOTE: In this study, fresh frozen human brain tumor tissues were taken for the lysate preparation. The protocol can be used for any fresh frozen tissue with some changes depending on the type of tissues (soft or hard tissues) and cellular complexity of the tissues.</li>
	<li>After that, spin the tube to settle the tissue and carefully remove the PBS using a pipette. Perform another PBS wash if there are still traces of blood left in the tissue.</li>
	<li>Add 300 &#181;L of urea lysis buffer (8 M urea, 50 mM Tris pH 8.0, 75 mM NaCl, 1 mM MgCl2) and protease inhibitor cocktail (PIC) as per the manufacturer&#39;s protocol. 
	NOTE: The volume of the lysis buffer should be enough to grind the tissue during the sonication process and to suspend what is extracted. Too little lysis buffer may result in inefficient tissue lysis, while too much lysis buffer will dilute the protein lysate.</li>
	<li>Place the tube on ice and sonicate the tissue at an amplitude of 40% for 2.5 min with pulse cycles of 5 s (ON/OFF, respectively).</li>
	<li>Add zirconium beads to the tubes and homogenize the tissue using a bead beater for 90 s with 5 min incubation on ice. Repeat this step twice.</li>
	<li>Once the tissue is adequately homogenized, incubate the tube on ice for 10 min.</li>
	<li>After incubation, centrifuge the sample at 6,018 x g for 15 min at 4 &#176;C to separate the cell debris from the supernatant.</li>
	<li>Collect the supernatant in the fresh labeled tube and store at -80 &#176;C as aliquots until further use.</li>
</ol>
2. Protein quantification and quality check of tissue lysates
<ol>
	<li>Quantify the protein concentration in the tissue lysate using Bradford&#39;s reagent as described in the Supplementary File 1.</li>
	<li>Following the protein quantification, run 10 &#181;g of tissue lysate on a 12% SDS-PAGE gel to check the quality of the lysate. 
	&#8203;NOTE: Further downstream processing must be carried out only for the lysates clearing the quality checks.</li>
</ol>
3. Enzymatic digestion of proteins
NOTE: The steps for enzymatic digestion are shown in Figure 1a.
<ol>
	<li>For digestion, take 50 &#181;g of proteins and add ddH2O to make up the volume to 20 &#181;L.</li>
	<li>Now, prepare 20 mM Tris (2-carboxyethyl) phosphine (TCEP) from the stock (0.5 M TCEP) by adding 0.8 &#181;L from stock to the protein lysate to reduce the disulfide bonds in the proteins and incubate the sample at 37 &#176;C for 60 min.</li>
	<li>Prepare 40 mM iodoacetamide (IAA) in ddH2O and add 1.6 &#181;L to alkylate the reduced cysteine residues. Incubate in the dark for 10 min at room temperature.</li>
	<li>Add dilution buffer containing 25 mM Tris pH 8.0 and 1 mM CaCl2 in a 1:8 ratio to dilute the urea concentration to less than 1 M in the sample. At this point, check the pH. 
	NOTE: If using trypsin as a digestion enzyme, make sure the concentration of urea is less than 1 M.</li>
	<li>To perform digestion, add trypsin at an enzyme/substrate ratio of 1:50. Incubate the tubes at 37 &#176;C in a shaking dry bath for 16 h for overnight digestion. 
	NOTE: The trypsin enzyme is a highly reactive protease that is prone to self-digestion. Take extra care and perform the addition of trypsin swiftly over the ice.</li>
	<li>After 16 h of incubation, dry the digested peptides in a vacuum concentrator.</li>
</ol>
4. Desalting of digested peptides
NOTE: To perform the desalting of peptides, use C18 stage tips.
<ol>
	<li>Activate the C18 stage tip by adding 50 &#181;L of methanol. Centrifuge the tip at 1,000 x g for 2 min at RT. Discard the filtrate collected at the bottom of the tube. Repeat twice.</li>
	<li>Add 50 &#181;L of acetonitrile in 0.1% formic acid to wash the stage tip. Centrifuge the tube at 1,000 x g for 2 min at RT. Discard the filtrate collected at the bottom of the tube. Repeat this step twice.</li>
	<li>Add 50 &#181;L of 0.1% (v/v) FA to equilibrate the column. Again, perform the centrifugation at 1,000 x g for 2 min at RT and discard the filtrate.</li>
	<li>Reconstitute the dried digested peptides in 50 &#181;L of 0.1% formic acid. 
	NOTE: Avoid air bubble formation inside the stage tips while passing the sample. The stage tips should not be completely dried during the centrifugation step, as drying can lead to peptide loss.</li>
	<li>Add the reconstituted peptides into the activated stage tip and pass the sample through the stage tip by centrifugation at 1,000 x g for 2 min. Repeat this step at least four times. Store the flow-through at 4 &#176;C.</li>
	<li>To wash the sample, add 50 &#181;L of 0.1% (v/v) formic acid. Repeat the centrifugation step and discard the filtrate.</li>
	<li>For the elution of peptides, add 50 &#181;L of 40% (v/v) ACN in 0.1% formic acid (v/v) and pass it through the stage tip by centrifugation. Collect the filtrate in a fresh tube. Repeat the step with 50% and 60% ACN in 0.1% formic acid and collect the filtrate in the same fresh tube.</li>
	<li>Dry the desalted peptides collected in the fresh tube using a vacuum concentrator. 
	&#8203;NOTE: The dried desalted peptides are ready to be injected, or it can be stored at -20 &#176;C for 6 months. For long-term storage (&#62;6 months), store the peptides at -80 &#176;C.</li>
</ol>
5. Quantification of desalted peptides
<ol>
	<li>Reconstitute the dried desalted peptides in 0.1% FA.</li>
	<li>Wipe the photometric measurement plate with lint -free tissue using 70% ethanol.</li>
	<li>Use 2 &#181;L of 0.1% FA to set the blank.</li>
	<li>Add 2 &#181;L of reconstituted samples onto the plate in replicates.</li>
	<li>Place the plate in the spectrophotometer and measure the absorbance at 205 nm and 280 nm.</li>
	<li>Calculate Molar Absorptivity (&#949;) using the following formula: 
	&#949;&#160;= 27 / [1 - 3.85 * A280 / A205] 
	NOTE: Molar absorptivity (&#949;) is a measure of the probability of the electronic transition or how well a species absorbs the particular wavelength of radiation that is being incident on it. The value of &#949; should be in the range of 31 mL mg-1cm-1 to 33 mL mg-1cm-1. If the value does not fall in the range, this indicates that the samples are not properly digested.</li>
	<li>Calculate the peptide concentration in &#181;g/&#181;L using the following formula: 
	Concentration of peptide = Net OD (205) / 0.051 * &#949;</li>
</ol>
6. Label-free quantitation (LFQ) of the digested peptides
NOTE: For label-free quantitation, use the LC and MS parameters mentioned in the Supplementary File 2. A high coverage data was obtained when three biological replicates of the same type of the sample were run in the mass spectrometer.
<ol>
	<li>Liquid chromatography setup
	<ol>
		<li>After the quantification of desalted peptides, take 2 &#181;g of peptides in a vial and make up the volume to 10 &#181;L using 0.1% FA. The concentration of desalted peptides will be 200 ng/&#181;L.</li>
		<li>Open the auto-sampler of the liquid chromatography system (see Table of Materials) and place the vial inside the autosampler.</li>
		<li>Use 0.1% (v/v) FA to equilibrate the pre-column and analytical column.</li>
		<li>Take 1 &#181;g of desalted digested peptide from the vial and load it onto the column.</li>
		<li>Set the LC gradient according to the sample complexity. In this experiment, LC gradient was used for 120 min for label-free quantitation of the tissue samples.</li>
	</ol>
	</li>
	<li>MS setup: Before optimizing any proteomics assays, perform a quality control check of the instrument by monitoring some peptides of Bovine Serum Albumin (BSA) using any software for system suitability and analyzing coverage of BSA (Figure 2A,B). The acquisition parameters were set into the instrument using the MS data acquisition software (see Table of Materials).
	<ol>
		<li>Open the software, double click on Instrument Set Up and select the template from peptides-ID with default parameters.</li>
		<li>Set the MS parameters using Supplementary File 2 and Save it as a new method.</li>
		<li>Now, open the software to fill the sample details; double click on the Sequence Setup, and fill in the details such as sample type, sample name, file save location, instrument method file, the volume of injection, and position of the sample.</li>
		<li>Once all the information is filled, select the row and start the Run.</li>
	</ol>
	</li>
</ol>
7. Label-based quantitation (iTRAQ) of digested peptides
NOTE: Label-based quantification can be performed using different isobaric labels such as iTRAQ or TMT reagents, etc. Here, iTRAQ 4-plex was used for the labeling of digested peptides from three tissue samples. The procedure of iTRAQ 4-plex labeling is mentioned below.
<ol>
	<li>Labeling of digested peptides using iTRAQ reagents. 
	NOTE: In this experiment, peptides from three tissue samples are used. From each tissue sample, 80 &#181;g of digested peptides are taken in four tubes for labeling with iTRAQ reagents (114, 115, 116, and 117) (see Supplementary File 3 for the detailed experimental parameters).
	<ol>
		<li>Before using the iTRAQ reagent, bring each vial of the reagent to room temperature (approximately 5 min). Give a brief spin of approximately 30 s to bring the solution at the bottom of each vial. 
		NOTE: Make sure that in each vial, 10-15 &#181;L solution should be present.</li>
		<li>For iTRAQ labeling, reconstitute the dried peptides in 20 &#181;L of dissolution buffer provided in the iTRAQ labeling kit.</li>
		<li>Reconstitute the labels by adding 70 &#181;L of ethanol from the vial provided in the kit and mix the solution for 30 s and spin it for 10 s. 
		NOTE: It is advisable that all the steps be carried out as per the manufacturer&#39;s instructions.</li>
		<li>Add the homogeneously mixed iTRAQ labels (114, 115, 116, and 117) to their respective tubes containing peptide samples and allow for the labeling reaction to take place.</li>
		<li>Mix the components of each tube by vortexing the tube for 30 s, and then spin the tube for 10 s to bring the mixture back to the bottom of the tube. 
		NOTE: Check the pH of the solution using pH paper. pH should be greater than 8; if not, add up to 10 &#181;L of the dissolution buffer to adjust the pH.</li>
		<li>Incubate each tube at room temperature for 90 min. At the end of the reaction, quench any excess unbound label in the tube by adding MS grade water.</li>
		<li>Incubate the tubes at room temperature for 30 min to 1 h.</li>
		<li>Once the incubation is over, transfer all the labeled contents into a single tube and dry the labeled peptides in a vacuum concentrator. 
		NOTE: A similar labeling procedure can be followed for TMT labeling.</li>
	</ol>
	</li>
	<li>Liquid chromatography setup
	<ol>
		<li>Reconstitute the samples in 0.1% formic acid, open the autosampler of nano LC, and place the samples inside the autosampler. Use the parameters mentioned in Supplementary File 2 for LC setup.</li>
		<li>Set the LC gradient according to the complexity of the sample. LC gradient of 180 min was used in this experiment for label-based quantitation (iTRAQ) of the tissue samples. 
		NOTE: For less complex samples, short gradient can efficiently separate most peptides. However, if the sample is very complex, use a longer gradient for better separation of peptides.</li>
	</ol>
	</li>
	<li>MS setup for iTRAQ technique
	<ol>
		<li>Set up all the MS parameters for label-based quantitation in the same way as used for the label-free quantitation except for the collision energy, which was set to 35% for MS/MS fragmentation in the label-based quantitation.</li>
	</ol>
	</li>
</ol>
8. Data analysis
<ol>
	<li>Analyze the raw (MS/MS spectrum) files obtained from LC-mass spectrometer using a commercially available analysis software (see Table of Materials). 
	NOTE: The Human Reference Proteome database from Uniprot (UP000005640) comprising 71,785 proteins sequences was used to obtain protein identities using Sequest HT and Mascot (v2.6.0) search engines. The parameters for label-free quantitation and label-based quantitation are described in Supplementary File 4.</li>
</ol>

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S., 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=A+timely+shift+from+shotgun+to+targeted+proteomics+and+how+it+can+be+groundbreaking+for+cancer+research.">A timely shift from shotgun to targeted proteomics and how it can be groundbreaking for cancer research.</a> Frontiers in Oncology. 7, 13 (2017).</li><li>Ray, S., 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=Proteomic+technologies+for+the+identification+of+disease+biomarkers+in+serum:+advances+and+challenges+ahead.">Proteomic technologies for the identification of disease biomarkers in serum: advances and challenges ahead.</a> Proteomics. 11 (11), 2139-2161 (2011).</li><li>Chen, E. I., Yates, J. 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The authors have nothing to disclose.

Tissue proteomics of biological samples enables us to explore new potential biomarkers associated with different stages of disease progression. It also explains the mechanism of signaling and pathways associated with disease progression. The described protocol for tissue quantitative proteomics analysis provides reproducible good coverage data. Most of the steps have been adapted from the manufacturer&#39;s instructions. In order to obtain high-quality data, the following steps are most crucial. Hence, extra care should ...

Proteomics technologies have the potential to enable the identification and quantification of potential candidate markers that can aid in the detection and prognostication of the disease1. Recent advancements in the field of mass spectrometry have&#160;accelerated clinical research at the protein level. Researchers are trying to address the challenge of complicated pathobiology of several diseases using mass spectrometry-based proteomics, which now offers increased sensitivity for protein identification and quantification2. Accurate quantitative measurement of proteins is crucial to comprehend the dynamic and spatial cooperation among proteins in healthy and diseased individuals3; however, such analysis on a proteome-wide scale is not easy.
One major limitation of proteomic profiling of clinical specimens is the complexity of biological samples. Many different types of samples have been investigated to study the disease proteome, such as cell lines, plasma, and tissues4,5. Cell lines are widely used as models in in vitro experiments to mimic different stages of disease progression. However, one major limitation with cell lines is that they easily acquire genotypic and phenotypic changes during the process of cell culture6. Body fluids such as plasma could be an attractive source for biomarker discovery; however, due to the highly abundant proteins and dynamic range of protein concentration, plasma proteomics is a bit more challenging7. Here, peptides originated from the most abundant proteins can suppress those derived from the low abundant proteins even if the mass/charge ratio is the same6. Although there have been advancements in the depletion and fractionation technologies in the last few years, getting good coverage still remains a major limitation of plasma proteomics8,9. The use of tissues for proteomic investigation of disease biology is preferred as tissue samples are most proximal to the disease sites&#160;and offer high physiological and pathological information to provide better insights into the disease biology10,11.
In this manuscript, we have provided a simplified protocol for the quantitative proteomics of tissue samples. We have used a buffer containing 8 M urea for the tissue lysate preparation as this buffer is compatible with mass spectrometry-based investigations. However, it is mandatory to clean the peptides to remove salts before injecting them into the mass spectrometer. One important point to remember is to reduce the urea concentration to less than 1 M before adding trypsin for protein digestion as trypsin exhibits low activity at 8 M urea concentration. We have explained two approaches of quantitative global proteomics: label-based quantification using iTRAQ (isobaric tags for relative and absolute quantification) and label-free quantification (LFQ). The iTRAQ-based quantitative proteomics is mainly used for comparing multiple samples varying in their biological condition (e.g., normal versus disease or treated samples). The approach utilizes isobaric reagents to label the N-terminal primary amines of peptides12. The iTRAQ reagents contain one N-methyl piperazine reporter group, a balancer group, and one N-hydroxy succinimide ester group that reacts with N-terminal primary amines of peptides13. Digested peptides from each condition are labeled with a particular iTRAQ reagent. Following the labeling, the reaction is stopped and labeled peptides from different conditions are pooled into a single tube. This combined sample mixture is analyzed by mass spectrometer for identification and quantification. After the MS/MS analysis, reporter ion fragments with low molecular masses are generated and the ion intensities of these reporter ions are used for the quantification of the proteins.
Another approach, label-free quantification is used to determine the relative number of proteins in complex samples without labeling peptides with stable isotopes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile (MS grade)</td>
			<td>Fisher Scientific</td>
			<td>A/0620/21</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>HiMedia</td>
			<td>TC194-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fischer Scienific</td>
			<td>BP510-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid (MS grade)</td>
			<td>Fisher Scientific</td>
			<td>147930250</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>1149-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (MS grade)</td>
			<td>Fisher Scientific</td>
			<td>Q13827</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Fischer Scienific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (MS grade)</td>
			<td>Fisher Scientific</td>
			<td>A456-4</td>
			<td></td>
		</tr>
		<tr>
			<td>MS grade water</td>
			<td>Pierce</td>
			<td>51140</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline</td>
			<td>HiMedia</td>
			<td>TL1006-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche Diagnostics</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Merck</td>
			<td>DF6D661300</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma</td>
			<td>646547</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Merck</td>
			<td>648310</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (MS grade)</td>
			<td>Pierce</td>
			<td>90058</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Bio-Rad</td>
			<td>5000205</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Merck</td>
			<td>MB1D691237</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypersil Gold C18 column</td>
			<td>Thermo</td>
			<td>25002-102130</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Gilson</td>
			<td>F167380</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage tips</td>
			<td>MilliPore</td>
			<td>ZTC18M008</td>
			<td></td>
		</tr>
		<tr>
			<td>Zirconia/Silica beads</td>
			<td>BioSpec products</td>
			<td>11079110z</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bead beater (Homogeniser)</td>
			<td>Bertin Minilys</td>
			<td>P000673-MLYS0-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader (spectrophotometer)</td>
			<td>Thermo</td>
			<td>MultiSkan Go</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Eutech</td>
			<td>CyberScan pH 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe Sonicator</td>
			<td>Sonics Materials, Inc</td>
			<td>VCX 130</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Drybath</td>
			<td>Thermo</td>
			<td>88880028</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Fusion mass spectrometer</td>
			<td>Thermo</td>
			<td>FSN 10452</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano LC</td>
			<td>Thermo</td>
			<td>EASY-nLC1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum concentrator</td>
			<td>Thermo</td>
			<td>Savant ISS 110</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proteome Discoverer</td>
			<td>Thrermo</td>
			<td>Proteome Discoverer 2.2.0.388</td>
			<td></td>
		</tr>
	</tbody>
</table>

comprehensive workflow of mass spectrometry-based shotgun proteomics of tissue samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

We have used two different approaches for discovery proteomics: label-free and label-based proteomics approaches. The protein profile of tissue samples on SDS-PAGE showed the intact proteins and could be considered for proteomic analysis (Figure 2A). The quality control check of the instrument was monitored via system suitability software and it showed the day-wise variation in the instrument performance (Figure 2B). We observed 91% sequence coverage of...

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Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

comprehensive-workflow-mass-spectrometry-based-shotgun-proteomics

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JoVE Journal

Biology

5.0K Views.  Indian Institute of Technology Bombay. The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Video: Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

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 ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...