News & Featured Publications
Analyze Time-Course Studies with 2D DIGE
May 22, 2023
A recent study in Blood Advances used our 2D DIGE service to analyze protein profile changes of platelets (PLT) over time.
When blood is stored in blood banks, the characteristics of platelets may change based on time and storage conditions. In current practice, platelets are stored at 22 C for up to 7 days, but this may harm cell structure and function. Another storage method at 4 C reduces waste and better preserves function but decreases circulation time.
In the study, 2D DIGE followed by mass spectrometry was used to compare platelets from Day 1, Day 7 4 C, and Day 7 22C . The results showed 78 spots with differential protein & post-translational modification (PTM) levels between the storage conditions. Among them, 10 PLT proteins showed drastic difference at their PTM level. Notably, several tubulin isoforms were found to be significantly under-expressed at Day 7 at 4 C compared to 22 C. Consistently, authors showed that PLT α-tubulin acetylation is enhanced acutely by HDAC-6 inhibition.
The identified proteins represent potential biomarkers for storage-induced activation in platelets and provide clues into the mechanism by which HDAC-6 regulates platelet conditions.
Comments: The challenge of finding reliable biomarker is not only to identify changes at the protein level, but also at PTM level, and the hardest to confirm these findings using an independent approach. The data here showed once again our 2D DIGE/MS platform could fulfill all these objectives.
2D DIGE vs 2D Gel
April 17, 2023
2D gel electrophoresis is an effective method of separating protein components by PI (isoelectric point) in IEF (1st dimension) and molecular weight in SDS PAGE (2nd dimension).
2D DIGE is an improved version of standard 2D gel. Instead of staining after running the gel, 2D DIGE uses CyDyes to label up to 3 different samples prior to running the gel. This platform thus offers several advantages:
1. If using 2D gel to compare two samples, running two separate gels is required and there will be gel-to-gel variation. In 2D DIGE, because up to 3 samples are run on the same gel, this variation is eliminated and the cost is also lower.
2. CyDye labeling has a sensitivity 250x that of Coomassie and 5x that of silver staining. This allows extremely high accuracy, spot resolution, and reproducibility.
3. Standard size 2D gels sometimes lack proper separation and can overlook low-abundance proteins. Our 2D DIGE is large-format, allowing a wide dynamic range that can resolve over 5000 proteins and detect low-abundance proteins, large proteins and small peptides.
Following are some applications of 2D DIGE that are impossible to achieve with 2D gel:
- Comparing protein profiles of three samples with no gel-to-gel variation
- Comparing protein profiles of >=4 samples by cross-gel analysis with accurate adjustment of gel-to-gel variation
- Assessing the HCP coverage of a drug / antibody, by running an HCP and an antibody western blot on the same 2D DIGE
- Visualizing and quantifying proteins with differential PTM expression
- Analyzing specific binding partners in immunoprecipitated (IP) proteins by filtering out un-specific bindings
Full Service: Immuno-Precipitation Proteomics
March 20, 2023
Because IP can be performed with non-cross linking or cross linking antibody, it is sometimes a challenge to decide the right proteomics approach for different IP methods. Applied Biomics offers two approaches to study IP samples:
- 2D DIGE / Mass Spec: compare protein profile of IP samples and identify only pulldown proteins with high resolution
- NanoLC – MS/MS: identify all proteins in the IP products and quantify their abundance
IP with non-cross linking antibody: The products will contain low-abundance pulldown proteins in the presence of high-abundance antibody / antigen proteins, which can pose a challenge. Because 2D DIGE offers high resolution of these low abundance proteins, it is the ideal approach for this IP method.
The following example shows an overlay image of a Control (Green labeled) and a Test (Red labeled) IP sample. One can clearly see that most proteins (in yellow) overlapping between Control and Test are from antigen and IgG. Only a handful of spots (in red and green) show the differential protein levels.
In this case, our 2D DIGE/Mass Spec platform offers unique advantages:
- High resolution: Well-resolved pull-down (target) proteins in the presence of abundant proteins
- High specificity: Filter out un-specific binding by including control sample
- Low cost: 3 samples in 1 gel; only ID the target spots (fewer than 10 spots in general)
- Fast turnaround: 5-7 days
IP with cross linking antibody: The products are much cleaner, containing mainly the associated pulldown proteins without any antibody proteins. Since NanoLC-MS/MS works best for these low/moderate complexity samples, it is the ideal approach for this IP method.
In addition, we can also perform IP experiments following our standard protocol or customer’s recommended protocols.
Elucidate Biotoxicity Mechanisms using 2D DIGE and MS
February 21, 2023
Spliceostatin is a well-known splicing inhibitor, but its mechanisms and phytotoxic effects in plants are still relatively understudied. A recent study in Frontiers used our 2D DIGE and Mass Spectrometry service to study how splicing inhibition affects the plant proteome. The project identified several differentially expressed proteins between samples treated with Spliceostatin C (SPC) and control.
Many of these proteins were located in the chloroplast, consistent with previous findings showing that this region is involved in the plant stress response. In addition, the presence of many proteins at a lower-than-expected molecular weight suggested that proteolysis was caused by SPC during this process. The phenotypic effects of SPC treatment, such as root inhibition and stomata impairment, were also reflected by the protein profile. Overall, the results help clarify the signaling pathways in response to SPC treatment and confirm its strong phytotoxic activity.
Comments: The data here showed again how powerful our 2D DIGE/MS platform is to quickly identify important protein markers from complex proteomes. More importantly, 2D images showed that many of these proteins migrate to a different location from their theoretical position, reflecting the protein profile changes at all 3 following aspects:
- Protein levels
- Post translation modifications (PTMs)
- Protein degradations or cross linking
Notably, #2 and 3 would be difficult to assess by LCMSMS or other proteomics techniques.
2023 New Year’s Promotion
January 3, 2023
New Year’s promotion: 10% Off Protein Identification (Offer Ends 02/03/2023)
Happy 2023! We would like to show our appreciation for choosing us to serve your proteomics needs. From January 3 to February 3, we are offering 10% off protein identification of 2D gel spots or 1D gel bands.
Take advantage of this limited-time discount to kick start your research for the new year. This offer applies to all our loyal existing customers as well as new customers.
*Terms: Only applies for Protein ID by standard MALDI-TOF/TOF, not LC-MS/MS. Cost of running 2D DIGE gel not covered. Does not apply to previous orders. Applied Biomics’ standard service terms and conditions apply.
Identify Critical Phospho-Sites using our Phospho-Site identification service
November 17, 2022
A recent study in eLife used Applied Biomics’ Phospho-Site identification service to identify critical phospho-sites of A-kinase anchoring protein 12 (AKAP12) involved in liver fibrosis.
Activation of hepatic stellate cells (HSCs) causes liver fibrosis by releasing extracellular matrix. Although previous work has shown an association between the phosphorylation of AKAP12 and HSC activation, the specific phosphorylation sites remain largely unknown.
Identification of phosphorylation-site involves enriching phosphorylated peptides followed by nanoLCMSMS. The presence of phospho-sites is confirmed by the presence of a MSMS peak showing the loss of phosphate (circled red).
From the MSMS spectra, multiple AKAP12 phosphorylation sites in activated HSC samples were identified and listed in the following table:
Interestingly, if these phosphor-sites were edited by CRISPR, the ability of AKAP12 to inhibit HSC activation is restored. The authors showed that AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling, and liver injury in mice.
Comments: This publication has clearly shown the significant role that phosphorylation could play. However, it is very challenging to study phosphorylation and other post translational modifications (PTMs) due to the very low level of these PTMs in vivo. In this project, we enriched the phosphorylated peptides and used the mass spectrometry settings for PTM detection, which enhanced detection sensitivity significantly.
If you are interested in a similar study, feel free to contact our support team with a brief description of your project. Our scientists will reach out to you shortly.
Track HCP profile during bioprocess
October 18, 2022
Applied Biomics offers comprehensive HCP analysis to detect and quantify HCP profiles during the purification process:
1. Qualitative Analysis: First, customers can visualize HCP compositions, modifications and fragmentations in a high resolution, large 2D image. This cannot be achieved by ELISA, LC-MSMS or other platforms.
2. Quantitative Analysis: Next, the HCPs can be analyzed quantitatively as following:
- Number of total detected spots in each sample
- Number of unique and overlapping spots between samples
- HCP quantitation: quantify individual HCP or total HCP in each sample.
- Spot map: Below is an example with 2-fold cutoff comparing CHO_S1 vs. CHO_S2. Red, green and yellow indicate spots with increased>2-fold, decreased>2-fold and within 2-fold, respectively
Please view here for a detailed example
3. Wide range of applications:
We have further optimized our proprietary sample preparation protocols for all major organisms used for generating recombinant therapeutic proteins. This assay can achieve 0.1 ng/spot sensitivity, equivalent to 1 PPM with 0.1 mg total protein loading. Quantitation of fluorescent signal allows accurate quantitation of the HCP and DS.
4. Cost-Effective
Lastly, each assay simultaneously tests and compares up to 3 samples, offering great saving of cost.
Featured Publication: Kinase Substrate Mapping Using Phospho-Proteomics Platform
September 19, 2022
A recent publication in Microbiology Spectrum used our PhosphoProteomics platform to identify important kinase substrates forProtein Kinase K (PknK) in Mycobacterium tuberculosis.
Pknk plays a key role in regulating the growth of M. tuberculosis. The PknK knock out strain showed increased survival under metabolic stress such as carbon and nitrogen starvation signals, compared to WT strains.
To identify Pknk substrates, authors compared the global phosphoproteomic profiles of WT and phosphorylation-defective PknK strains. Both the phosphorylation and protein level of each protein was assessed. Among thousands of proteins, only 22 spots showed positive phosphorylation level in the WT but not the Pknk-defective strain, as well as protein level change.
Further mass spectrometry of the 18 spots identified Pknk substrates such as Rho transcription terminator and several critical enzymes involved in metabolism and signal transduction.
Together with other findings, this study further established the Pknk signal transduction network and its central role for mycobacterial survival.
Comments: It is challenging to study protein phosphorylation due to the low phosphor-level and lack of effective separation between phosphor-protein and native protein. Our PhosphoProteomics platform addresses both these issues through fast screening and quantification of phospho-proteins with high sensitivity and resolution. The authors in this publication were able to quickly narrow down their targets to 22 protein spots among thousands of proteins. More importantly, proteins identified using our platform were validated by transcriptome and in vivo analysis.
Featured Service: Choosing the right sample type for HCP Antibody Coverage
August 17, 2022
It is critical to detect and measure HCPs in therapeutic products. However, a common drawback in many assays is the inability to detect protein modifications and degradations.
Our HCP Antibody Coverage platform can detect HCP composition, protein modifications, and degradations with high sensitivity and consistency – making it advantageous to ELISA, AAE and standard 2D Western blot. These features can greatly improve the purification steps during manufacturing and purity analysis of the final drug substance (DS).
While high quality HCP antibody is critical for optimal HCP coverage, it is equally important to determine the correct sample type: host cell culture supernatant, cell pellets, or others.
Here, we tested HCP antibody coverage of the same antibody against CHO cell supernatant (CHO-S) and CHO cell pellets (CHO-P). A striking difference is observed: 97.1% for CHO-S and 70.0% for CHO-P.
If you are interested, feel free to contact our support team with a brief description of your project. Our scientists will reach out to you shortly.
Featured Publication: Tissue Barriers
July 20, 2022
A recent publication in Tissue Barriers used our 2D DIGE / Mass Spec platform to identify key proteins in toxin-induced cell death.
Cholix is an exotoxin which induces cell death after crossing the epithelium. After comparing the protein profile of Cholix magnetic capture samples to the whole cell membrane (control) by 2D DIGE, 45 proteins showed significant differences. All 45 proteins were identified by mass spectrometry. One of the most over-expressed proteins in Cholix magnetic capture samples was GRP75 (spot 16).
GRP75, a heat shock protein that plays an important role in vesicles, was found to interact with Cholix during its crossing of the epithelium. Following endocytosis, GRP75 limits the delivery of Cholix to lysosomes.
More importantly, the binding of GRP75 to Cholix was further confirmed by 2 other platforms: IP and ELISA.
Comments on the data: With the fast development of proteomics technologies, researchers are often facing the challenge of identifying ‘real’ biomarkers that can be validated by other platforms. The findings in this paper proved again that 2D DIGE is a reliable platform to analyze complex proteomes and identify biomarkers.
If you are interested, feel free to contact our support team with a brief description of your project. Our scientists will reach out to you shortly.
Featured Service: Improved HCP antibody coverage protocol
June 15, 2022
Blue: 83 spots not detected by antibody
Anti-CHO HCP antibody coverage: 97.3%
One main challenge in performing HCP antibody coverage by 2D Western blot is the unspecific antibody binding, which causes high background and inaccuracy in quantitation. To address this issue, Applied Biomics has performed extensive internal studies testing different conditions.
With our recent improvements to the protocol, we were able to achieve:
- Significantly lower background
- Overall higher HCP antibody coverage
Featured Publication: bioRxiv
May 18, 2022
Study protein distribution within cells using our mass spectrometry service. A recent bioRxiv paper used Mass Spectrometry to identify proteins in various sections of a giant cell. Findings revealed that 30% of the Stentor proteome has polarized localization.
News Release: Applied Biomics Offers Qualitative and Quantitative Host Cell Protein (HCP) Profiling Analysis
February 19, 2020
From Business Wire:
Applied Biomics, Inc., a leading Proteomics service provider, adds a HCP profiling analysis using their well established high resolution, large format 2-Dimensional Differential In-Gel Electrophoresis (2D DIGE) platform. This assay can be used to monitor HCP contamination during the purification process thus helps to improve purification strategy, specifically:
1) Visualize and overlay the HCP images between different samples
2) Quantitate HCP content by spot counting and PPM for each sample
3) Quantitate the purity of product or drug substance (DS) by spot counting and PPM
4) Quantitatively compare HCP content by fold-change and overall similarity between samples
