Serum Proteomics

Our Serum & Plasma proteomics service is the ideal platform for serum / plasma biomarker discovery and validation. It can resolve & quantitate thousands of serum proteins even WITHOUT immuno-depletion. Our improved serum protocol has successfully identified low abundant serum protein markers for therapeutics and diagnostics..

Why 2D DIGE but not LC-MS/MS for serum/plasma proteomics? Because 2D DIGE can generate much better serum proteomics data at a lower cost:

• Fast screening: 2D DIGE physically separates low abundant proteins from abundant proteins by both Mw and pI, without depleting serum abundant proteins
• High sensitivity: Fluorescent labeling improves detection of low-abundance proteins.
• Fast turnaround: Results in 5–7 days.
• Cost-effective: Compares up to 3 samples in a single test;

Serum Proteomics Applications

• Suitable sample types: serum, plasma, media
• Biomarker Discovery: Identify disease-associated protein biomarkers.
• Disease Diagnosis: Detect protein signatures associated with specific diseases.
• Treatment Response: Monitor proteomic changes before and after therapy.
• Drug Discovery: Identify potential therapeutic targets and mechanisms of action.
• Disease Progression: Track protein changes across disease stages.

Serum Proteomics Workflow

• Remove Serum abundant proteins (optional)
• Fluorescent sample labeling
• 2D gel electrophoresis
• Image acquisition & image analysis
• Quantitative analysis
• Automated spot picking
• LC-MS/MS protein identification
• Pathway analysis & validation analysis (optional)

Serum Proteomics Deliverables

• Image report
  • High-resolution 2D proteome images for each sample
  • Overlay images comparing two samples
  • Mark differentially expressed spots
• Quantitation report
  • Protein abundance ratios for differentially expressed spots
  • Statistical P-value (if applicable)
• Protein ID report
  • Protein Level: Protein identity, abundance, sequence coverage, and confidence metrics
  • Peptide Level: Matched peptide sequences, masses, and confidence metrics

Serum Proteomics Study Design & Examples

• A. Improved serum 2D DIGE without immuno-depletion
• B. Improved serum 2D DIGE combined with immuno-depletion of abundant proteins

Pros and Cons of Immunodepletion: Immunodepletion has been used to enhance the detection of low abundance plasma proteins. However, immunodepletion (i) removes proteins associated with the abundant proteins, (ii) causes protein degradations, and (iii) introduces variations, thus compromising data quality. Research has discovered that even with immunodepletion, low abundance proteins still remain undetectable using LC-MS/MS platform.

A. Improved Serum 2D DIGE Without Immuno-depletion

Example 1: Human Serum Sample

The left, middle and right image shows the human serum proteome using standard protocol, Applied Biomics (ABO) improved protocol with pH3-10, and pH4-7, respectively. The ABO protocol clearly reveals much more low abundant serum proteins than the standard protocol.

Standard 2D Protocol (pH 3-10)

ABO pH 3-10 Protocol

ABO pH 4-7 Protocol

Example 2: Preclinical Trial Mouse Serum Sample

Four pairs of mouse serum samples were collected before (Control) and after 4 different treatments (Treated). 2D DIGE was performed using Applied Biomics’ improved serum protocol. The 4 Control (red) /Treated (green) overlay images showed the differences between Control and Treated samples. The circled spots represent very low abundant proteins which are up-regulated due to drug treatment. The 3-D view of the same spot is displayed in the right panel. The data shows that ABO protocol significantly improves the detection of low abundant proteins in the serum samples.

B. Improved Serum 2D DIGE Combined With Immuno-depletion Of Abundant Proteins

The Challenge of Biomarker Discovery from Plasma/Serum

Transferrin: 76 kDa
Fibrinogen: 65 kDa (a), 51 kDa (g)
a1-antitrypsin: 56 kDa
IgG: 150 kDa
HSA: 67 kDa
IgA: 180 kDa
IgM: 750 kDa
Transferrin: 79 kDa
a2-Macroglobulin: 160 kDa
Haptoglobin: 43 kDa
alpha 1-acid glycoprotein: 23 kDa
APO A-I, APO A-II: 23 kDa

Protein dynamic range: 10 magnitude of difference in abundance

We offer the following serum abundant protein immuno-depletion services:

1. Human Serum: Albumin/IgG depletion
2. Human Serum: IgY-12 depletion (12 most abundant human plasma proteins: Human Serum Albumin, IgG, Fibrinogen, Transferrin, IgA, IgM, Haptoglobin, a2-Macroglobulin, a1-Acid Glycoprotein, a1-Antitrypsin and HDL (Apo A-I & Apo A-II).
3. Mouse Serum: Albumin/IgG depletion
4. Rat Serum: Albumin/IgG depletion
5. Monkey Serum: Albumin/IgG depletion

Example: Human plasma with and without IgY-12 Immuno-depletion (pH4-7)

Plasma Without Immuno-Depletion

Plasma With Immuno-Depletion