<ol>
	<li>Fu, Q., 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=Highly+Reproducible+Automated+Proteomics+Sample+Preparation+Workflow+for+Quantitative+Mass+Spectrometry.">Highly Reproducible Automated Proteomics Sample Preparation Workflow for Quantitative Mass Spectrometry.</a> Journal of Proteome Research. 17 (1), 420-428 (2018).</li><li>Lehmann, 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=Clinical+mass+spectrometry+proteomics+(cMSP)+for+medical+laboratory:+What+does+the+future+hold?.">Clinical mass spectrometry proteomics (cMSP) for medical laboratory: What does the future hold?.</a> Clinica Chimica Acta. 467, 51-58 (2017).</li><li>Abbatiello, S. E., 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=Large-Scale+Interlaboratory+Study+to+Develop,+Analytically+Validate+and+Apply+Highly+Multiplexed,+Quantitative+Peptide+Assays+to+Measure+Cancer-Relevant+Proteins+in+Plasma.">Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma.</a> Molecular &amp; Cellular Proteomics. 14 (9), 2357-2374 (2015).</li><li>Kuster, B., Schirle, M., Mallick, P., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scoring+proteomes+with+proteotypic+peptide+probes.">Scoring proteomes with proteotypic peptide probes.</a> Nature Reviews Molecular Cell Biology. 6 (7), 577-583 (2005).</li><li>Carr, S. A., 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=Targeted+peptide+measurements+in+biology+and+medicine:+best+practices+for+mass+spectrometry-based+assay+development+using+a+fit-for-purpose+approach.">Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.</a> Molecular &amp; Cellular Proteomics. 13 (3), 907-917 (2014).</li><li>Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+role+of+computers+and+software+in+mass+spectrometry+-+SEQUEST+and+20+years+of+tandem+MS+database+searching.">Pivotal role of computers and software in mass spectrometry - SEQUEST and 20 years of tandem MS database searching.</a> Journal of the American Society for Mass Spectrometry. 26 (11), 1804-1813 (2015).</li><li>Nilsson, T., 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=Mass+spectrometry+in+high-throughput+proteomics:+ready+for+the+big+time.">Mass spectrometry in high-throughput proteomics: ready for the big time.</a> Nature Methods. 7 (9), 681-685 (2010).</li><li>Van Eyk, J. E., Sobhani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+Medicine.">Precision Medicine.</a> Circulation. 138 (20), 2172-2174 (2018).</li><li>Hoofnagle, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+Generation,+Quantification,+Storage,+and+Handling+of+Peptides+Used+for+Mass+Spectrometry-Based+Assays.">Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.</a> Clinical Chemistry. 62 (1), 48-69 (2016).</li><li>Wijayawardena, B., Fu, Q., Johnson, C., Van Eyk, J. E., Kowalski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Reproducible+Automated+Proteomics+Sample+Preparation+on+Biomek+i-Series.">Highly Reproducible Automated Proteomics Sample Preparation on Biomek i-Series.</a> Technical note, Beckman Coulter Life Science. Document # AAG-4890APP02. 19, (2019).</li></ol>

Sample processing for mass spectrometry requires protein denaturation, reduction and alkylation to block cysteines, and trypsin digestion to cleave proteins into peptides. Each chemical or enzymatic reaction needs to be initiated at a designated time and performed at a controlled temperature, and every step in the process involves multiple liquid transfer steps where experimental variation can be introduced. Automated sample processing provides a solution to this dilemma. Presently available liquid handling systems have the capability to transfer reagents into 96-well plates with an accuracy and precision of less than 5%, depending on the head and tip type used, and to incubate samples, with shaking if desired, under controlled temperatures ranging from 14 &#176;C to 70 &#176;C. We used an automated liquid handler to process plasma for SRM assays in a 96-well format.
There are many thousands of protease cleavage sites within the complex mixtures of proteins in serum, plasma, and other biological samples; and each of these proteins has unique properties affecting cleavage site accessibility and the stability of the resulting peptides. It is, therefore, impossible to design a sample processing procedure that is optimal for every protein. The best alternative is to be as consistent as possible.
To achieve consistency, we optimized each pipetting step performed by the automated liquid handler. We first considered the volume required and constraints imposed by the liquid&#8217;s type (plasma, aqueous, or organic) and corresponding properties (viscosity, cohesion, and volatility), hardware (workstation pipetting-head and plate-grabbing arms), and labware. We then varied the speed and trailing air gap for aspiration, the speed and blowout volume for dispensing, and the force and duration of mixing, while incorporating a tip touch after aspiration and/or dispensing, if needed, to eliminate liquid adhering to the outside of the tips (Figure 13, Supplemental Table 1). A unique SIL peptide, pre-screened for stability, was spiked into each reagent to make it possible to monitor the precision of each liquid handling step. After optimization, the process CVs for the majority of the transitions were less than 10%, demonstrating good reproducibility with the automated workstation (Figure 9, Figure 10, Figure 11 and Figure 12).
The automated workflow presented here provides for consistent enzymatic digestion with improved reproducibility and throughput compared to manual methods (Figure 9, Figure 10). This approach promises to improve the accuracy and reliability of biomarker discovery and validation by mass spectrometry.

Mass spectrometry (MS) -based protein and peptide quantification is increasingly being applied as a bioanalytical tool for plasma analysis in basic research and clinical laboratories2,3. The requisite instrumentation and informatics have advanced rapidly as MS has become the method of choice for quantifying proteins or modified proteins by targeting specific peptide sequences due to its ability to quantify hundreds of peptides in a single MS run4. Sample preparation serves as the foundation for any proteomic analysis. Prior to MS analysis, the proteins in a biological sample are typically denatured, reduced, alkylated, digested into tryptic peptides, and desalted5. Alkylation blocks cysteines to prevent uncontrolled modifications and ensure all cysteines have the same mass. Next, trypsin is added to digest proteins into peptides. Each of these steps requires optimization, and the entire process is traditionally performed manually, allowing for the introduction of analytical errors.
The traditional biomarker development pipeline consists of two main processes: shotgun proteomics for global protein discovery to create an in-depth protein inventory6 and targeted proteomics for verification and validation aimed at high-precision and high-throughput protein quantitation7. Regardless of the MS approach, sample preparation is the same and centers on enzymatic cleavage of proteins into a peptide mixture. As proposed by Van Eyk and Sobhani8, having a method that allows for accurate, precise, and reproducible analysis for both discovery and targeted assays is desired to effectively move discovery biomarkers to clinical implemented assays. To do this, automation of sample preparation will be helpful and provide the ability to increase the efficiency to high-throughput analysis. Manual methods typically introduce analytical errors beyond acceptable limits specified by the Food and Drug Administration (FDA) in the revised Bioanalytical Method Validation Guidance, which includes biomarkers and diagnostics2,9. The development of a fast, highly accurate, and hands-free MS protein sample preparation workflow will be necessary to facilitate biomarker research, which requires preparing and analyzing thousands of biological samples. MS sample preparation has many steps where errors can be introduced, and the process is tedious and time-consuming.
In our improved method, an automated workstation was programed to perform all necessary plasma sample preparation procedures in a total of 6 steps (Figure 1) to ensure: 1) accurate liquid transfers; 2) the reaction is initiated and stopped at a consistent time; 3) the reaction is performed at a controlled temperature (i.e., incubator); and 4) the reaction has uniform mixing for all reactions. We also implemented adding exogenous quality control proteins and internal standards (stable isotope-labeled peptide standards) to ensure a quality and reproducible throughput of LC-MS-based protein and peptide quantification in a 96-well format. Including reagent preparation, hours of work time and a total of 1,000,000 pipetting steps would be required to process 96 samples in individual tubes. Automation reduces the hands-on time and the number of human interactions involved.

<table>
	<tbody>
		<tr>
			<td>A Pooled healthy human plasma</td>
			<td>Bioreclamation Inc.</td>
			<td></td>
			<td>human plasma tested in the manuscript</td>
		</tr>
		<tr>
			<td>Acetonitrile, HPLC Grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>A998SK1</td>
			<td>LC MS/MS solvent</td>
		</tr>
		<tr>
			<td>B-Galactosidase Recombinant from E.Coli</td>
			<td>Sigma-Aldrich</td>
			<td>G3153-5MG</td>
			<td>exogenous control proteins.</td>
		</tr>
		<tr>
			<td>Biomek i7 Automated Workstation</td>
			<td>Beckman Coulter, Inc.</td>
			<td></td>
			<td>The proteomic sample preparation workstation: Biomek automated workstations are not intended or validated for use in the diagnosis of disease or other conditions</td>
		</tr>
		<tr>
			<td>Biomek i-Series Tips 230&#181;L Non-Sterile</td>
			<td>Beckman Coulter, Inc.</td>
			<td>B85903&#160;</td>
			<td>Tips, i7 consumable</td>
		</tr>
		<tr>
			<td>Biomek i-Series Tips 90&#181;L Non-Sterile</td>
			<td>Beckman Coulter, Inc.</td>
			<td>B85881&#160;</td>
			<td>Tips, i7 consumable</td>
		</tr>
		<tr>
			<td>FG, Kit Trypsin TPCK</td>
			<td>Sciex</td>
			<td>4445250</td>
			<td>Trypsin used in digestion</td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear&#160;</td>
			<td>Bio-Rad</td>
			<td>#hsp9641</td>
			<td>Autosampler plate</td>
		</tr>
		<tr>
			<td>Octyl B-D-Glucopyranoside</td>
			<td>Sigma-Aldrich</td>
			<td>O9882-5G</td>
			<td>detergent used in digestion</td>
		</tr>
		<tr>
			<td>Polypropylene, 96-Round Deep Well Plates Sterile</td>
			<td>Beckman Coulter, Inc.</td>
			<td>267007</td>
			<td>Reagent and digestion plate</td>
		</tr>
		<tr>
			<td>Prominence UFLCXR HPLC system</td>
			<td>Shimadzu, Japan</td>
			<td></td>
			<td>High flow LC sytem</td>
		</tr>
		<tr>
			<td>QTRAP 6500</td>
			<td>SCIEX</td>
			<td></td>
			<td>Mass spectrometer</td>
		</tr>
		<tr>
			<td>Water, HPLC Grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>W54</td>
			<td>LC MS/MS solvent</td>
		</tr>
		<tr>
			<td>Xbridge BEH30 C18 2.1mm x 100mm</td>
			<td>Waters</td>
			<td>186003564</td>
			<td>LC MSMS column</td>
		</tr>
	</tbody>
</table>

a plasma sample preparation for mass spectrometry using an automated workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Watch this Scientific Journal Video about A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation at JoVE.com

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

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Biochemistry

9.8K Views.  Cedars Sinai Medical Center. Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Video: A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

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

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

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

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

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

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

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

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

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

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

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

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

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

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

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

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

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

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

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

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

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

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

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

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

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

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

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