Proteomic toolbox for autoimmunity research
Introduction
Autoimmune diseases are associated with significant mortality and morbidity; hence, early diagnosis and treatment is absolutely important. Traditional methods for diagnosis may sometimes be inadequate since they may be invasive or relatively non-specific. Fortunately, over the past 10 years, proteomic technologies have experienced a revolution since the introduction of MALDI-TOF, a powerful mass spectrometer for protein sequencing. In pace with this, other novel technologies have also grown fairly quickly, such as protein arrays, phospho-proteomics and quantitative proteomic methods such as ICAT and iTRAQ. Although proteomics is still a relatively young technology, its application in the field of autoimmune diseases has already shown great potential in uncovering novel biomarkers and therapeutic targets, as reviewed [1], [2], [3], [4], [5].
Proteomics has its roots in analytical chemical techniques for protein separation, and has evolved through three stages: 1. 2-D gel based approaches; 2. Non-quantitative, non-gel based approaches, like 2D LC-MS-MS; 3. Quantitative, non-gel based approaches including ICAT and iTRAQ. In addition, a variety of “biased” or targeted antibody-coated or antigen-coated protein microarrays have also become increasingly popular. In this review, we briefly survey current proteomic technologies and their applications in autoimmunity.
Section snippets
Gel based approaches: 2D-gel-MS
2D-gel electrophoresis, the workhorse of proteomics research over the past decade, has been quite widely used in autoimmunity. One of the earliest reports focused on the study of rheumatoid arthritis. Using the 2D-gel/MS, S100A9, a small calcium-binding protein, was highlighted as a potential marker for RA [6]. Oates et al have used the same approach to identify protein spots that can be used to predict ISN/RPS renal pathology class and chronicity in SLE [7]. Giusti et al. have studied the
Non-quantitative, non-gel based approaches: 2D-LC MS
A commonly used non-gel based method for protein separation and identification is two-dimensional (2D) online liquid chromatography-mass spectrometry (LC/MS). This technology involves the elution of digested peptides from a strong cation exchange column followed by reversed phase separation, and identification by MS. Liao et al. have studied the synovial proteome from RA patients with erosive versus non-erosive arthritis using 2D LC-MS, and have uncovered 3 calcium binding proteins of the S100
Non-quantitative, non-gel-based approaches: SELDI-TOF MS
Surface enhanced laser desorption/ionization time-of-flight MS (SELDI-TOF MS) allows protein chips with either chemically or biochemically modified surfaces to bind different subsets of proteins for identification by MS. Though rarely used in autoimmunity research, Mosley et al. have employed SELDI-TOF MS to discriminate the urinary proteomes between active and inactive lupus nephritis allowing them to correctly distinguish 92% of the inactive and active SLE patients [12].
Quantitative, non-gel based approaches: iTRAQ
iTRAQ (Isobaric Tagging for Relative and Absolute Quantitation) is a relatively new proteomic technology. Protein quantification is achieved by comparing the peak area of 4 reporter ions used. Recently, Liu et al. have used the iTRAQ approach to identify ~ 20 differentially expressed proteins in the spinal cords of rats with experimental autoimmune encephalomyelitis, including I2PP2A [13]. Likewise, we have applied iTRAQ to detail the urine proteins in lupus nephritis, and have uncovered > 50
Targeted proteomics: Antigen coated array
In contrast to the above approaches, targeted array-based proteomic platforms begin with a finite set of proteins or antibodies, and seek to ascertain if subsets of these candidates may be useful in distinguishing diseased samples from normal controls. Such a focused array has been used to study autoantibody specificities. The first autoantigen array designed to detect and characterize autoantibodies was developed by Joos et al. [14]. Later on, a larger-scale autoantigen array was developed by
Targeted proteomics: Cytokine/chemokine antibody array
Cytokine/chemokine antibody arrays are multiplexed, sandwich-based ELISA assays designed to determine the concentration of multiple cytokines within one experiment. Such an array has been used to detect the levels of 62 proteins in urine from mice with immune mediated nephritis [17]. Interestingly, increased urinary levels of VCAM-1, sTNFR-1, CXCL16 and P-selectin, correlated well with the degree nephritis in mice and patients with SLE [17], [18]. Using a modified version of this approach,
Targeted proteomics: Multiplexed immunoblots
Immunoblot is a classical immunological technique based on antigen-antibody interactions. Recently, this tool has empowered the discovery of “pathogenic”/“target” autoantigens in autoimmunity. First, proteins extracted from biological tissues of interest (skin, kidney, brain, spleen, lymph node, thymus, etc) are separated by electrophoresis. Next, serum from patients can be used as the primary antibody to recognize and identify “target” antigens/proteins on these blots. Recently, Lefranc etc
Targeted proteomics: Single cell proteomics
In addition to the above approaches, there is another relatively small-scaled proteomic method called “single cell proteomics”. To date, there are 2 major technologies built upon single cell proteomics; one is based on flow cytometry [22], the other is “probed isoelectric focusing (PIF)” [23]. Phospho flow cytometry is a very new technique, where one stains selected cell surface markers, and simultaneously stains intracellular antigens in the same cell. Surface staining helps identify the cell
Current challenges and future directions
As previously reviewed [1], [2], [3], [4], [5], [31], [32], [33], proteomics is still at its infancy, with each method having its advantages and disadvantages. As we look to the future, 3 challenges need to be addressed. First, though 2D-gels have emerged as one of the most popular proteomic approaches, it suffers from relatively poor reproducibility. To surmount this, future 2D-gel approaches must be developed to allow for automation and high throughput. An example of this is a modification
Take-home messages
- •
Unbiased proteomic technologies have evolved through three stages: 2-D gel based approaches, non-quantitative, non-gel based approaches, like 2D LC-MS-MS, and thirdly, quantitative, non-gel based approaches including ICAT and iTRAQ.
- •
All 3 of the above classes of unbiased proteomic platforms have been executed in a variety of autoimmune diseases.
- •
In addition, a variety of “biased” or targeted antibody-coated or antigen-coated protein microarrays have also become attempted in the study of
References (34)
- et al.
Proteomic analysis of the saliva: a clue for understanding primary from secondary Sjogren's syndrome?
Autoimmun Rev
(2008) - et al.
The laboratory approach to the diagnosis of autoimmune diseases: is it time to change?
Autoimmun Rev
(2007) - et al.
Autoimmunity in the era of genomics and proteomics
Autoimmun Rev
(2006) - et al.
Prediction of urinary protein markers in lupus nephritis
Kidney Int
(2005) - et al.
Proteomic surveillance of autoimmunity in Behcet's disease with uveitis: selenium binding protein is a novel autoantigen in Behcet's disease
Exp Eye Res
(2007) - et al.
Single cell profiling of potentiated phospho-protein networks in cancer cells
Cell
(2004) - et al.
Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach
Mol Cell Proteomics
(2006) - et al.
Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research
Mol Cell Proteomics
(2004) - et al.
Global investigation of p53-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded tagging
Mol Cell Proteomics
(2004) - et al.
A dataset of human liver proteins identified by protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry
Mol Cell Proteomics
(2004)