Part of this work was supported by NIH grant HL090630 (ISK).

1. Preparation steps for the peel-blot technique
NOTE: Preparation requires for the following items to be placed adjacent to each other.
<ol>
	<li>Stack two pieces of 20-25 &#181;m pore size filter paper (see Table of Materials).</li>
	<li>Place an ~8 cm x 10 cm long paraffin film adjacent to the filter paper with the paper facing up.</li>
	<li>Place a piece of submicron filter paper on top of two additional pieces of #4 filter paper (Figure 1-1).</li>
	<li>Position the anti-capillary forceps with the bent leg facing up and the straight leg facing down. Then, select a 600-mesh grid with the smooth/shiny side up. Place the forceps on a Petri dish or other raised surface to avoid contact of the clean grid with other items.</li>
	<li>Remove the paper from the paraffin film and pull the sides (~5 mm) to create a small rim. Pipette two 150 &#181;L drops of 4% trehalose solution ~1-2 cm from one short side of the paraffin film and ~1 cm apart on the paraffin film.</li>
	<li>On a separate bench or on the floor, pour liquid nitrogen in a small polystyrene foam container (see Table of Materials). 
	CAUTION: Receive training for proper handling using gloves and a face shield in order to avoid frost bite.</li>
	<li>Place a small cryo-EM grid container in the liquid nitrogen and cover the polystyrene foam container with lint-free wipes.</li>
</ol>
2. Placing the carbon film onto the grid
<ol>
	<li>Use Dumont #5 forceps (see Table of Materials) to float of the carbon film from the mica by touching one side of the mica at a slight angle downwards (~20&#176;-40&#176;) on one of the drops of trehalose solution and slowly let the water tension lift off the carbon film by moving the mica downwards. Release the mica once the carbon film floats on the surface of the droplet.</li>
	<li>Visually inspect the carbon film to avoid using carbon with damage in the form of fractures.</li>
	<li>Insert the grid held by the anti-capillary forceps at an angle (~20&#176;-30&#176;) into a trehalose drop at a position adjacent to, and then centered under the carbon film. Pick up the carbon film (Figure 1-2).</li>
	<li>Keep the grid in the same orientation and slowly lower it onto the surface of the second drop of trehalose without breaking the surface tension of the drop to remove unnecessary carbon film that may have wrapped around the rim of the grid to the rough side.</li>
</ol>
3. Separation of multi-layered 2D crystals into single layers
<ol>
	<li>Rotate the anti-capillary forceps (see Table of Materials) and place them on a Petri dish with the carbon side of the grid facing down (Figure 1-3).</li>
	<li>Pipette 1.3 &#181;L of the sample into the slight trehalose meniscus on top of the grid. Pipette the solution ~8-10 times to mix and distribute the trehalose and sample on both sides of the grid.</li>
	<li>While incubating the solution for 1 min, pipette one or more 1.7 &#181;L drops of trehalose solution onto the paraffin film.</li>
	<li>Rotate the grid back into its original position with the carbon film facing up.</li>
	<li>Use the anti-capillary forceps to press the grid onto the submicron filter paper (Figure 1-4). Once the solution has been absorbed by the filter paper and the carbon film is lying flat, wait for 3s before lifting the grid and moving it vertically onto the 1.7 &#181;L drop of trehalose solution (Figure 1-5). 
	NOTE: Repeat this step multiple times for highly stacked samples, or the grid is prepared for vitrification in the next steps.</li>
	<li>Blot the grid on the two pieces of filter paper, and then lift the grid from the filter paper (Figure 1-6). After approximately 13 s, depending on humidity and sample buffer, plunge the grid into the liquid nitrogen in the small Styrofoam container and place the grid in the cryo-EM grid container or transfer it directly for cryo-EM screening or data collection. 
	NOTE: In order to rapidly optimize the protocol, negatively stain the peel-blotted grid at this step rather than plunging it into liquid nitrogen. Negatively stain the sample by applying 3 &#181;L of 1% uranyl acetate to the grid, incubate the grid for 30 s, and blot it with a torn piece of 20-25 &#181;m pore size filter paper. Grids of 2D crystals vitrified in trehalose, tannin, or glucose are routinely hand-plunged for vitrification as trehalose, tannin, and glucose prevent the formation of crystalline ice at rates used for hand-plunging2.</li>
</ol>

<ol>
	<li>Johnson, M. C., Uddin, Y. M., Neselu, K., Schmidt-Krey, I. 2D crystallography of membrane protein single-, double- and multi-layered ordered arrays. Methods in Molecular Biology. 2215, 227-245 (2021).</li>
	<li>Ku&#776;hlbrandt, W., Downing, K. H. Two-dimensional structure of plant light harvesting complex at 3.7 &#197; resolution by electron crystallography. Journal .of Molecular Biology. 207&#160;(4), 823-828 (1989).</li>
	<li>Nannenga, B. L., Gonen, T. MicroED: a versatile cryoEM method for structure determination. Emerging Topics in Life Sciences. 2&#160;(1), 1-8 (2018).</li>
	<li>Martynowycz, M. W., Zhao, W., Hattne, J., Jensen, G. J., Gonen, T. Collection of continuous rotation microED data from ion beam-milled crystals of any size. Structure. 27&#160;(3), 545-548 (2019).</li>
	<li>Gonen, T. et al. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature. 438&#160;(7068), 633-638(2005).</li>
	<li>Gonen, T. The collection of high-resolution electron diffraction data. Methods in Molecular Biology. 955, 153-169 (2013).</li>
	<li>Wisedchaisri, G., Gonen T. Phasing electron diffraction data by molecular replacement: strategy for structure determination and refinement. Methods in Molecular Biology. 955, 243-272 (2013).</li>
	<li>Gipson B., Zeng X., Zhang Z. Y., Stahlberg, H. 2dx-user-friendly image processing for 2D crystals. Journal of Structural Biology. 157&#160;(1), 64-72 (2007).</li>
	<li>Biyani, N. et al. Focus: The interface between data collection and data processing in cryo-EM. Journal of Structural Biology. 198&#160;(2), 124-133 (2017).</li>
	<li>Righetto, R., Stahlberg, H.Single particle analysis for high-resolution 2D electron crystallography. Methods in Molecular Biology. 2215, 267-284 (2021).</li>
</ol>

The authors do not have competing financial interests or other conflicts of interest.

The peel-blot is a powerful method to overcome stacking and thickness of multi-layered 2D crystals and similar samples for cryo-EM data collection and image processing. A decision on the use of the peel-blot will be based on biological parameters of the sample and will also require assessment once a 3D cryo-EM model is available. The biological importance of contacts and potential flexibility of contact regions will be particularly important in this assessment.
The number of peel-blots will initially be established via observation after standard negative stain grid preparation, where excessive layers or thickness will require alternative strategies to standard cryo-EM grid preparation. Thin multi-layered crystals that are stacked in register may be directly used for microED3&#160;or FIB-milled in the case of thicker crystals4. The peel-blot will usually not be necessary for double-layered 2D crystals as layers can be processed individually or together, when the layers are in register5. It could potentially be beneficial for exposing both sides of membrane protein 2D crystals to ligands or inhibitors though, once in solution before peel-blotting, and once after the final peel-blotting step. In addition, reducing large, well-ordered double-layered 2D crystals may allow for electron diffraction6,7. Multi-layers, either induced or biologically occurring, of 2D crystals of membrane and soluble proteins will be the most likely targets for the peel-blot, even though the protocol can be applied to a range of specimens and serve to concentrate various samples for cryo-EM (Figure 4).
Initial testing with negative stain will dramatically reduce the time and cost to optimize the samples as freezing, preparation of cryo-EM sample holders, transfer to a cryo-EM, and use of a cryo-EM are not required. Only the drying time before the freezing step will need to be optimized separately. The drying time will be identical or very similar to the time used for drying a standard back-injection grid of the same sample1.
While 600-mesh grids generally result in good preservation of the carbon film, increased numbers of peel-blot iterations and thinner carbon film layers may lead to increased carbon film breakage (Figure 5). Stacked 2D crystals, liposomes, and layered samples of large arrays of soluble proteins can be successfully separated (Figures 2-4).
The sample is carefully evaluated by cryo-EM to ensure that adverse effects on sample quality are avoided. This may be achieved by image processing to compare images of stacked and unstacked crystals. Both for assessment purposes and to achieve the goal of a 3D model, image processing is conducted via the 2dx programs available in Focus8,9, which also implements single particle approaches to address lattice imperfections and protein heterogeneity with the possibility to dramatically increase upon the inherent quality of the original 2D crystals10.

The peel-blot technique was developed in order to separate stacked two-dimensional crystals of membrane proteins into single layers to obtain suitably thin samples for cryo-EM 2D and 3D data collection and facilitate image processing1. The protocol is similarly suitable for other types of multilamellar samples as well as for achieving high sample concentrations of either sample type on the grid without increasing the thickness in Z.
Reduction of sample layers to one layer is achieved by applying a combination of capillary pressure and shearing forces in a protocol that substantially extends on and modifies the back-injection method2. A first blotting step results in tight sandwiching and adherence to the carbon film and a grid bar on either side of the multi-layered sample during blotting on submicron rather than the 20-25&#181;m filter paper pore size in the back-injection method. The separation, or peeling, of an individual layer occurs upon forcing the separation of the sandwiched layers once the grid is pressed vertically onto a trehalose drop, forcing the carbon film away from the grid bar (Figure 1). Repositioning of the carbon film away from the original contact point with the grid bar occurs in the next step, when the grid is once again blotted on submicron filter paper. The number of iterations of these steps can be optimized for specific samples. Furthermore, the protocol can be used to increase sample concentrations while avoiding aggregation in Z. Due to the reliance on adherence to the grid bar and carbon film as well as forceful separation, this approach may not be suitable for highly fragile molecules such as certain delicate and/or unstable proteins and protein complexes.
The peel-blot technique neither requires specialized equipment nor expensive supplies. Applicability to specific samples will, however, require careful considerations of biological parameters. The decision is easier for 2D crystals as carbon film is required to ensure flatness, yet manipulation via the peel-blot technique may affect the biological integrity of the sample or crystalline order. Suitability of the peel-blot technique to single particles is restricted to and may be tested for those that require carbon film and are stable to manipulation. Specimens with critical biological interactions between layers may not be suitable for characterization with help of the peel-blot technique due to the disruption of such interactions.
The peel-blot technique (&#34;peel-blot&#34;) is most quickly optimized by testing and screening with negative stain or unstained samples by TEM at room temperature. Once the optimal number of peel-blot iterations have been identified, the time to control thickness before vitrification can be determined.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>600-mesh grids</td>
			<td>SPI</td>
			<td>2060-C-XA</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-capillary forceps</td>
			<td>Ted Pella</td>
			<td>510-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon to coat mica</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo-EM</td>
			<td></td>
			<td></td>
			<td>Cryo-EM grids may be screened at 120 kV or 200 kV.&#160; High-resolution data is collected at 300 kV.</td>
		</tr>
		<tr>
			<td>Dumpont #5 forceps</td>
			<td>Ted Pella</td>
			<td>5622</td>
			<td></td>
		</tr>
		<tr>
			<td>Grid box for cryo-EM storage</td>
			<td>Ted Pella</td>
			<td>160-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Kim Wipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mica</td>
			<td>Ted Pella</td>
			<td>56</td>
			<td>The mica is carbon-coated and cut into squares that are slighlty larger than a TEM grid.&#160; The carbon thickness may require optimization to avoid thin carbon that breaks easily upon multiple peel blots.</td>
		</tr>
		<tr>
			<td>Negative stain</td>
			<td></td>
			<td></td>
			<td>1-2% uranyl acetate is suitable for many samples.&#160; Other stains such as phosphotungstic acid can be substituted.</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene container</td>
			<td></td>
			<td></td>
			<td>Used for vitrifying the peel-blot grid. A polystyrene shipping container can be recycled for this purpose and lined with aluminium foil.</td>
		</tr>
		<tr>
			<td>Submicron filter paper</td>
			<td>MilliporeSigma</td>
			<td>DAWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>JEOL</td>
			<td>JEM-1400</td>
			<td>Any TEM operated at an accelerating voltage of 80-120 kV will be suitable for screening of negatively stained grids.</td>
		</tr>
		<tr>
			<td>Trehalose</td>
			<td></td>
			<td></td>
			<td>Prepare 4% trehalose solution.</td>
		</tr>
		<tr>
			<td>Whatman #4 filter paper</td>
			<td>MilliporeSigma</td>
			<td>WHA1004150</td>
			<td>This corresponds to the 20-25 &#956;m pore size filter paper in the protocol.</td>
		</tr>
	</tbody>
</table>

the peel-blot technique: a cryo-em sample preparation method to separate single layers from multi-layered or concentrated biological samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

A successful peel-blot experiment will result in single layers of samples at often high concentrations. It is to be expected that some regions in most grid squares will still contain multiple layers, especially at fewer numbers of iterations of the peel-blot steps. The majority of grid squares, however, will contain extensive regions of single layers as observed for 2D crystals of human leukotriene C4&#160;synthase (Figure 2) and liposomes (added in step 3.2, Figure 3). Additional peel-blot steps may need to be tested for samples that do not show the desired reduction in layers.
The most important indication of a successful peel-blot experiment will be the data quality and biological integrity observed following high-resolution cryo-EM data collection and image processing. Data interpretation will need to include careful consideration of potential damage or distortion to contact regions between layers.
Regions of the EM grid in which peel blotting has occurred typically display a higher concentration of sample than regions in which it has not occurred. This apparent concentrating effect is caused by the adherence of the multilamellar sample to both the carbon film and the grid bar surfaces, followed by additional adherence to both surfaces during the peel-blot preparation as they repeatedly separate, reposition slightly, and again make contact. The result is that the different layers of the original multi-lamellar sample are spread apart over a larger lateral area (Figure 3 and Figure 4). These regions can even contain larger 2D crystals or membranes. Both phenomena are easily observed and compared in areas where a &#34;peel zone&#34; is adjacent to an area not affected by the peel-blot step (Figure 3). The images in Figures 2-4&#160;were collected under low-dose conditions at an accelerating voltage of 120 kV with a transmission electron microscope (see Table of Materials).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64099/64099fig01.jpg" /> 
Figure 1: Schematic representation of the peel-blot technique. The first two steps (1-2&#160;and 1-3) and the last step (1-6) of the peel-blot technique are based on the back-injection method2&#160;and the intermediate steps follow the peel-blot protocol (modified from Johnson et al.1). In step 1, (A) two pieces of 20-25 &#181;m pore size filter paper are stacked, (B) adjacent to a piece of paraffin film with two drops of trehalose solution and (C) a piece of submicron filter paper on two additional pieces of filter paper. In step 2, (A) the carbon film is floated off on the first trehalose droplet and (B) picked up with anti-capillary forceps, after which it is touched to the surface of the second droplet (not shown) without breaking the surface tension to remove excess carbon film from the periphery. In step 3, (A) the forceps holding the grid are rotated by 180&#176; (the carbon film side of the grid is now facing down) and (B) the sample is pipetted into the meniscus of the trehalose. In step 4, the grid has been rotated back to the original orientation with the carbon film facing up and the sample is slowly lowered onto the submicron filter paper. The grid is lifted up and in step 5 lowered vertically onto a 1.7 &#181;L drop of trehalose solution. Steps 4 and 5 are referred to as &#34;peel-blotting&#34; and can be iterated three or more times, depending on the amount of stacking. The number of iterations may need to be optimized for different samples. In step 6, (A) the sample is blotted on two layers of 20-25 &#181;m pore size filter paper, before (B) it is hand plunged into liquid nitrogen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64099/64099fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64099/64099fig02.jpg" /> 
Figure 2: A 2D crystal sample subjected to the peel-blot method. The region to the left of the arrow was largely reduced to single-layered 2D crystals in a contact region between the carbon film and grid bar that was peel-blotted and then shifted, while the region to the right of the arrow was not in a region affected by the peel-blot. The peel-blotted regions can easily be selected for and are observed on most of the grid. The 2D crystals shown are stacked and single-layer 2D crystals of human leukotriene C4&#160;synthase. The scale bar corresponds to 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64099/64099fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64099/64099fig03.jpg" /> 
Figure 3: Liposomes subjected to the peel-blot. The peel-blot can successfully be applied to interrupt the edges of collapsed liposomes and vesicles to result in regions with large, mostly single-layered phospholipid bilayers as observed in the lower half of the image. The upper half was not in a contact zone between the grid bar and the carbon film and thus contains complete liposomes with two phospholipid bilayers. The scale bar corresponds to 1 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64099/64099fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64099/64099fig04.jpg" /> 
Figure 4: The peel-blot footprint. The grid square of a 400-mesh grid shows the footprint (labelled &#34;B&#34;) of the grid bar and the resulting peel-blotted regions as well as the regions (labelled &#34;C&#34;) that were not in contact with a grid bar, or subjected to fewer peel-blot iterations. The scale bar corresponds to 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64099/64099fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64099/64099fig05.jpg" /> 
Figure 5: Blotting an EM grid prepared by back-injection on a submicron filter paper, using carbon film that is too thin. Images are single frames from a video (see Video 1) taken at (A) initial contact with the membrane, (B) 1000 ms after contact, and (C) 2000 ms after contact. The arrow indicates grid squares in which capillary pressure has ruptured the carbon film. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64099/64099fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: The grid is placed on the submicron filter paper for the peel-blot step. The carbon film is slowly and tightly suctioned onto the grid in the direction from the lower left to upper right, which tightly sandwiches sample between the grid bar and carbon film. The carbon film and thus individual sample layers are displaced in each subsequent peel-blot step, allowing for optimization with additional iterations for more highly layered samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64099/video1.mov" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples at JoVE.com

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

the-peel-blot-technique-cryo-em-sample-preparation-method-to-separate

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.2K Views.  Georgia Institute of Technology. The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

Video: The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

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TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

Cryo-EM and Single-Particle Analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...