Aliquoting of isobaric labeling reagents for low concentration and single cell proteomics samples

The introduction of isobaric tagging reagents enabled more accurate, high-throughput quantitative proteomics by enabling samples to be multiplexed. One drawback of these workflows is the relative expense of the proprietary chemical reagents, which is often only second to the expense of the instruments themselves. These highly reactive chemical tags are only commercially available in relatively large aliquots compared to the typical amounts of peptides analyzed in proteomic workflows today. Excess reagents are typically disposed of following labeling of small batches or within a few weeks of opening. We present a simple procedure to aliquot commercial isobaric tagging reagents and demonstrate the successful and high efficiency labeling of multiple samples over a period of six months. The samples presented herein were selected as the most diverse samples labeled by prepared aliquots from a single labeling reagent kit. We observe comparable labeling efficiency from 100 microgram to 100 picograms of peptide when labeling samples from both human digest standards, cancer cell lines prepared in-house and from cells directly obtained from organ donations, despite differences in cell type, lysis and digestion. No labeling experiment of whole human proteomics samples achieved less than 92% labeling efficiency over this period. When preparing phosphoproteomic samples from a cancer cell line digest at approximately 6 months from the date of the aliquoting procedure, we observed a decrease in labeling efficiency to approximately 86%, indicating we are approaching the end of the useful lifetime of these prepared aliquots. Over this period, we have effectively reduced the reagent costs of each labeling experiment to less than 10% of the predicted costs when following the manufacturer instructions for use and disposal. While aliquoting of reagents can be performed by hand, we provide a complete template for automatic aliquoting using an affordable liquid handling robot, including plans for 3D printing of parts we have found useful for streamlining this procedure.

NQR sensitive embedded signatures for authenticating additively manufactured objects

Automatic recognition of unique characteristics of an object can provide a powerful solution to verify its authenticity and safety. It can mitigate the growth of one of the largest underground industries—that of counterfeit goods–flowing through the global supply chain. In this article, we propose the novel concept of material biometrics, in which the intrinsic chemical properties of structural materials are used to generate unique identifiers for authenticating individual products. For this purpose, the objects to be protected are modified via programmable additive manufacturing of built-in chemical “tags” that generate signatures depending on their chemical composition, quantity, and location. We report a material biometrics-enabled manufacturing flow in which plastic objects are protected using spatially-distributed tags that are optically invisible and difficult to clone. The resulting multi-bit signatures have high entropy and can be non-invasively detected for product authentication using $$^{35}$$ 35 Cl nuclear quadrupole resonance (NQR) spectroscopy.

The impact of ligand binding based assays critical reagent characterization and storage

Biological critical reagents are the foundation of many bioanalytical methods and often chemically modified or conjugated with various chemical tags. As such, the quality and performance of these methods are inherently tied to the quality and stability of critical reagents. This article will outline recommendations for conjugated critical reagent development and characterization. Examples of the impact of regent quality will be discussed for the two common bioanalytical assays in support of drug development for biotherapeutics. Finally, a brief discussion of conjugated reagent stability and recommendations for storage and testing will be presented.

Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

<p></p><p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ </i>visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.</p><p></p>

Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

<p></p><p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ </i>visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.</p><p></p>

Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

<p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves <a></a><a>photon, electron or X-ray</a> based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a <a>chemical</a> tag into the protein <i>via</i> genetic code expansion technique. The method was developed and validated by imaging GFP in <i>E. coli</i> and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in <i>E. Coli</i> cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ</i> visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.<br></p>

Protocol of 4-plex N,N-Dimethyl Leucine Isobaric Labeling Based Proteome Quantitation by LC-MS/MS

Isobaric labeling is one of the wide-spread high throughput proteome quantification strategies, where enzymatically digested protein extracts from different experimental groups are labeled with chemical tags and yield identical mass shift for individual peptides in MS precursor scan, whereas quantitation can be achieved by comparing reporter ion ratios upon fragmentation in tandem MS scan. In addition to commercial tags such as TMT and iTRAQ, we developed 4-plex and 12-plex N,N-dimethyl leucine (DiLeu) tags as cost-effective alternatives with comparable performance 1,2. DiLeu labeling methods allow multiplexing of samples and enable comparative analysis of acetyl-CoA flux regulated proteomes from different mouse models in the same LC-MS runs, providing statistically reliable data that avoids run-to-run variations. This protocol describes steps including 40 hrs of sample preparation of lysis, tryptic digestion, tag labeling, SCX purification and high pH fractionation, as well as details about LC-MS/MS analysis and data processing. The protocol can be applied to other proteome quantitation systems.