News & Featured Publications

Effects of pyruvate kinase M (PKM2) Tyrosine Phosphorylation on bone cell differentiation

September 19, 2023

A publication in March 2023 used our nanoLC-MS/MS service to determine top candidates for phosphorylation. In the experiment, total cell lysate was prepared from IgSF11-treated culture, passed through phosphoprotein affinity columns, and separated by SDS-PAGE. After confirmation of tyrosine phosphorylation by Western Blot, the major phosphorylated band was excised for mass spectrometry analysis.

The nanoLC-MS/MS identified three candidates for phosphorylation: vimentin, tubulin beta 5, and PKM2. Importantly, data analysis identified phosphorylation at Y105 as a unique identifier of PKM2 in immunoglobulin superfamily 11 (IgSF11) activated proteins.

Further analysis showed that IgSF11 activates several tyrosine kinases, which phosphorylate PKM2, causing lower PKM2 activity. Conversely, IgSF11-deficient cells show higher PKM2 activity and defective osteoclast differentiation. The data indicated that PKM2 acts a metabolic switch in the development of bone cells.

Remarks: this publication shows that our mass spectrometry platform was able to identify a key phospho-protein as well as its specific phosphorylation site. More importantly, follow-up validation studies confirmed the significance of this protein and its phosphorylation in the context of bone cell differentiation.

Accurately detect and quantify Intact Protein Mass

August 17, 2023

Applied Biomics’ Intact Mass services offer accurate detection & quantitation of purified intact proteins. Using top-of-the-line Orbitrap Exploris technology, we have optimized from sample preparation to mass spectrometry scan settings for different kinds of proteins.

In an intact mass experiment, proteins are introduced into the mass spectrometer without digestion. Because each protein is different, mass spectrometry settings must be optimized. The mass spectrometer then separates the intact protein into components, producing a “charge envelope.” Typically there are multiple high-abundance m/z peaks, with the highest peak in the middle.

The raw data is then submitted to BioPharma Finder 5.1, which generates a “deconvoluted spectra.” The most abundant mass of the protein is shown as a dominant main peak, while other minor components are shown as smaller peaks. Both main and minor components will be quantified.

Data Report covers the following:

  1. MS Spectra: Protein fingerprint showing the peaks and charge states at various m/z
  2. Deconvoluted Spectra: Main peak & mass of the protein, along with smaller peaks of minor components
  3. Component-level data
  4. Charge state-level data

Accurately quantitate between samples with our LFQ service

July 10, 2023

Applied Biomics’ Label Free Quantitation (LFQ) services offer high-sensitivity detection and accurate quantitation of proteins in any protein sample. By combining optimized protocols and top-of-the-line Orbitrap Exploris Technology, we deliver high-quality data with fast turnaround and a low cost.

Label-free quantitation uses the intensities of spectra to compare relative & absolute abundance between samples. Due to the high sensitivity of Orbitrap Mass Spectrometers and sophisticated Proteome Discoverer software, this is a reliable approach with a more straightforward design than labeled methods.

The success of LFQ does not depend on labeling efficiency—a major challenge for labeled methods. In addition, LFQ is not constrained by the number of labeling reagents, allowing an unlimited number of samples to be compared. Finally, the protein coverage in LFQ is generally higher than in labeled approaches.

Our LFQ data report consists of the following:

  1. Protein abundance in each sample
  2. Confidence metrics
  3. Detailed protein information
  4. Protein functions, locations and pathways (if available in database)
  5. Peptide-level data

Phosphoprotein Profiling shows how CD44-dependent phosphorylation facilitates parasite invasion

June 14, 2023

CD44 is an important host factor for the invasion of malaria parasite but its mechanism is unknown. A recent study used our phosphoprotein profiling platform to compare both protein & Phosphoprotein profiles between WT RBCs and RBCs treated with Erythrocyte Binding Antigen-175 (EBA-175), a CD44 binding partner.

In the study, two sets of 2D DIGE experiments were performed:

  1. WT total protein vs EBA-175-treated total protein
  2. WT Phosphoprotein vs EBA-175-treated Phosphoprotein

The data revealed 50 spots with >1.3 fold protein level change, and 26 spots with >2 fold increased Phosphorylation in the EBA-175-treated sample. After the Phospho ratio was adjusted based on protein levels, the six spots with highest adjusted-Phospho ratios were identified by mass spectrometry.

Table showing the protein ID of six spots with highest adjusted-phospho ratios.

Taken as a whole, the proteomics data not only provides clues into potential proteins involved in the EBA-175 induced signaling pathway, but demonstrates that EBA-175 stimulation leads to increased Phosphorylation of several RBC proteins.

Comments:

Post translational modifications (PTMs) play a key role in cell signaling pathways. However, the low level of PTMs makes it challenging to detect and quantify the PTM change in the complex proteome. Our Phosphoprotein profiling platform offers: visualization, fast screening, and quantitation of phospho-protein changes that no other platform can achieve.

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.

Miyazawa B, Trivedi A, Vivona L, Lin M, Potter D, Nair A, Barry M, Cap AP, Pati S. Histone deacetylase-6 modulates the effects of 4°C platelets on vascular endothelial permeability. Blood Adv. 2023 Apr 11;7(7):1241-1257.

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.

Significant storage-induced changes in PLT posttranslational protein modification

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.

2D DIGE vs 2D Gel experimental procedure

Following are some applications of 2D DIGE that are impossible to achieve with 2D gel:

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:

  1. Protein levels
  2. Post translation modifications (PTMs)
  3. 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

Happy New Years 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.

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