Biomarkers in inflammatory bowel diseases: Current status and proteomics identification strategies

Table 1 Known common inflammatory bowel disease biomarkers

Biomarker	Specificity	Usability
Serum biomarkers
ASCA	39%-79% of CD patients positive, 5%-15% UC patients[41-43]	14%-18% of controls tested positive, limiting the diagnostic value[44]
pANCA	20%-85% of UC patients positive, 2%-28% of the CD patients[41,42,45]	32% of controls tested positive, limiting the diagnostic value[44]
CRP	Marker for acute inflammation	Cannot differentiate CD from UC. However, usable for monitoring disease state[48-50]
Fecal biomarkers
Calprotectin	Sensitive marker for intestinal inflammation[8,17,40]	Cannot differentiate CD from UC. Used to monitor disease state[17]
Lactoferrin	Can distinguish active IBD from inactive IBD and irritable bowel syndrome[60]	Unspecific for CD and UC. However, usable for monitoring disease state[60]

ASCA: Anti-Saccharomyces cerevisiae antibodies; ANCA: Anti-neutrophil cytoplasmic antibody; IBD: Inflammatory bowel diseases; UC: Ulcerative colitis; CD: Crohn’s disease; CRP: C-reactive protein.

Table 2 Proteomics biomarker candidate studies and main findings

Ref.	Sample	Analysis	Findings and perspectives
Barcelo-Batllori et al[66], 2002	In vitro colon epithelial cells and purified epithelial cells from UC and CD patients	2D-PAGE protein quantitation, and in-gel digestion and MALDI-TOF MS and Western blot protein identification	The enzyme indoleamine-2,3-dioxygene was more abundant in cells from CD and UC patients compared to normal mucosa. Tryptophan and arginine metabolism may play a role in the IBDs
Hardwidge et al[67], 2004	Human Caco-2 intestinal epithelia cells before and after infection with E. coli	ESI LC-MS protein identification and quantitation, Western blot verification	125 proteins more abundant and 139 proteins less abundant after infection, some related to innate immune responses. These proteins might be relevant to look for in future biomarker studies
Hsieh et al[68], 2006	Colonic biopsies from UC, nonspecific infectious colitis patients and controls	2D-PAGE protein quantitation, and in-gel digestion and MALDI-TOF MS protein identification	6 proteins were found to be more abundant in UC and 13 less abundant. The result indicates that mitochondrial dysfunction might be involved in UC the etiology. Four biomarker candidates were identified, however, they require validation
Shkoda et al[69], 2007	Intestinal tissue cells purified from patients suffering from CD, UC, and colon cancer	2D-PAGE protein quantitation, and in-gel digestion and MALDI-TOF MS and Western blot identification	Proteins associated with signal transduction, stress response and energy metabolism were differently abundant in inflamed and non-inflamed tissue. 32% of the differentially regulated proteins were involved in energy metabolism
Meuwis et al[10], 2007	Serum from UC and CD patients	SELDI-TOF MS m/z signal profiling, MALDI-TOF MS and Western blot protein identification	Successful in differentiating CD from UC patients with a sensitivity of 85% and a specificity of 95% from several m/z signals. Four biomarker candidates were identified, all known acute inflammatory markers, limiting the diagnostic value. However, the feasibility of serum biomarker studies was demonstrated
Nanni et al[71], 2007	Serum from UC, CD patients and healthy controls	Solid-phase bulk protein extraction, MALDI-TOF MS signal profiling	Able to separate the three groups with 97% prediction results. The signals were not identified, but the feasibility of serum biomarker studies was demonstrated
Meuwis et al[70], 2008	Serum from responding and non-responding CD patients to infliximab	SELDI-TOF MS signal profiling, MALDI-TOF MS, Western blot and ELISA protein identification	Able to predict responders with a sensitivity of 79% and a specificity of 80%. Increased amount of PF4 was associated with non-response to infliximab with MS but not ELISA, so usability of PF4 as a biomarker seems limited
Nanni et al[72], 2009	Intestinal epithelial cells from CD patients and healthy controls	1D-PAGE and in-gel digestion, ESI LC-MS protein identification and quantitation	Proteins more abundant in CD patients include several proteins involved in inflammation processes, and less abundant include Annexin A1, involved in the anti-inflammatory action. Follow-up research is required to assess the feasibility of the biomarker candidates
Hatsugai et al[73], 2010	Peripheral blood mononuclear cells from UC and CD patients, and healthy controls	2D-PAGE quantitation, and in-gel digestion and MALDI-TOF MS protein identification	Successfully discriminated UC from CD based on seven differently present proteins, all associated with inflammation oxidation/reduction, the cytoskeleton, endocytotic trafficking and transcription.The biomarker candidates require validation using a larger number of patients, but seems promising
M’Koma et al[74], 2011	Mucosal and submucosal layers of samples originating from CC and UC patients	MALDI-TOF MS m/z signal characterization, no protein identification	Five m/z signals were detected in the submucosal layer, which could separate the two groups with an accuracy of 75 percent. The signals needs to be identified, however, the disease groups can be separated on basis of the mucosal and submucosal profiles
Presley et al[75], 2012	Microbes and human proteins at the intestinal mucosal-luminal interface from CD and UC patients, and healthy controls	Oligonucleotide ribosomal RNA fingerprinting, SELDI-TOF MS and MALDI-TOF MS identification	35% of the detected bacterial phylotypes were present in different amounts in the diseases, indicating the involvement of host-microbe interactions in IBD. The microbiome might prove useful as a target for therapy
Han et al[14], 2013	Colonic tissue biopsies of Korean IBD patients	ESI LC-MS protein identification with label-free quantitation	27 potential biomarkers were identified for UC, 37 biomarkers for CD and 11 proteins commonly associated with IBD. Three novel biomarkers were identified for active CD: Bone marrow proteoglycan, L-plastin and proteasome activator subunit 1. The biomarker candidates require validation, but might prove feasible as new diagnostic and therapeutic targets
Seeley et al[76], 2013	Histological tissue layers from UC and CC patients	MALDI-TOF MS m/z signal characterization, no protein identification	114 different m/z signals were found to be different between the two groups. The signals remain unidentified
Gazouli et al[77], 2013	Serum samples from responding and non-responding CD patients to infliximab treatment	2D-PAGE quantitation, and in-gel digestion and MALDI-TOF MS protein identification	15 differently abundant proteins between responders and non-responders to infliximab were identified.The biomarker candidates require further validation

IBD: Inflammatory bowel diseases; UC: Ulcerative colitis; CD: Crohn’s disease; MALDI-TOF MS: Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; 2D-PAGE: Two-dimensional polyacrylamide gel electrophoresis; ESI: Electrospray ionization; SELDI-TOF MS: Surface-enhanced laser desorption/ionization time of flight mass spectrometry; LC: Liquid chromatography.