In this issue: Proteomics – Clinical Applications 4/2008

In this issue of Proteomics you will find the following highlighted articles: MALDI and methyl groups take on lysosomal storage disease diagnosis Lysosomal storage disorders (LSD) result from the absence or loss of any of a wide variety of glycan‐processing enzymes that lead to accumulation of incompletely processed glyco‐molecules in lysosomes. Diagnosis of the particular type of LSD requires identification of the accumulated species, usually from urine. LSD diagnosis has been a target for many years, including use of GC/MS, TLC, NMR, HPLC, FACE and other techniques. Faid et al. take a simple approach that is quite successful and much less time consuming. After permethylating crude urine, a sample is first run on GC/MS to look for free sialic acid, an indicator of one set of diseases, then a cleaned up sample (C_1–8) is analyzed by MALDI/TOF to characterize glycoproteins and glycopeptides present. The glycans were further analyzed by sequential exoglucosidase digestion on the MALDI target. Sulfated glycocompound analysis requires a desulfation step prior to permethylation. Faid, V. et al., Proteomics Clin. Appl. 2008, 2, 528–542. Popular prostate problem receives piercing look Prostate cancer (PCa) is one of the most frequent male cancers. It is also one of the most frequently misdiagnosed cancers. Prostate specific antigen (PSA) has improved the accuracy somewhat but still generates 700 000 false positives requiring biopsies per year in the US. And very low PSA levels don’t assure freedom from PCa. In short, there is no good biomarker for PCa. Theodorescu et al. recognized that anything recovered from blood had a good chance of being at least partially degraded so they looked at first void urine as a more likely source of stable material. Applying capillary electrophoresis coupled to TOF‐MS, the authors developed an “informative” panel of eight peptides that distinguished between first void and mid‐stream samples and a PCa‐specific panel of 12 proteins that detected the disease. Incorporating the 12 proteins with age and free PSA, sensitivity was 91%, specificity 69%. Theodorescu, D. et al., Proteomics Clin. Appl. 2008, 2, 556–570. No candy in candidiasis Candida albicans is a unicellular yeast that is the third most common nosocomial agent in ICU’s, costing ～US$ 40 000 per incident. Early diagnosis of systemic candidiasis (SC) is crucial for successful control of the infection by antifungal therapies. Blood cultures are not fast and tissue biopsies are not sensitive but are the current “gold standards.” Pitarch et al. report here a serum protein marker for SC: anti‐Candida enolase (Eno1p) IgG. Comparing healthy with SC patients for antibodies against Candida cytoplasmic antigens, 15 antigens were recognized by the SC patients. The strongest response was to Eno1p which was the only antigen to produce a response in all 12 patients. The IgG was tested for its suitability as an antigen in Western and capture ELISA tests. Although the tests appear reliable for detecting SC, they were not good for prognosis. A considerable amount of work remains for full qualification of the test. Pitarch, A. et al., Proteomics Clin. Appl. 2008, 2, 596–618.     

Reliability of antibodies to Candida methionine synthase for diagnosis, prognosis and risk stratification in systemic candidiasis: A generic strategy for the prototype development phase of proteomic markers

To be able to diagnose systemic candidiasis (SC) and to predict outcomes in SC patients are still challenging tasks for physicians. Previous proteomic studies suggest that anti-Candida methionine synthase (Met6p) IgG antibodies may be a candidate marker for SC. To evaluate their reliability for diagnosis, prognosis and risk stratification in SC, we developed a generic prototype strategy for their measurement in SC. Receiver-operating-characteristic curve analyses revealed a high diagnostic accuracy for this prototype format, which was slightly better to that for the widely used Western blot assays. Multivariate logistic-regression models showed a positive association between serum anti-Met6p IgG antibody levels and SC risk that was independent from established SC risk factors and other baseline variables. After adjusting for and stratifying according to known prognostic factors, a significant trend toward a lower two-month mortality risk with increasing levels was evidenced in SC patients at presentation. We conclude that these antibodies may be useful in discriminating SC from non-SC patients and determining risk stratification in SC. These may also confer protection against SC and be valuable for the design of future immunotherapies. Furthermore, our prototype format has the potential to make impact on other infectious diseases, cancers, allergies or autoimmune disorders.     

Cover Picture: Proteomics – Clinical Applications 4/2008

In this issue of Proteomics you will find the following highlighted articles: MALDI and methyl groups take on lysosomal storage disease diagnosis Lysosomal storage disorders (LSD) result from the absence or loss of any of a wide variety of glycan‐processing enzymes that lead to accumulation of incompletely processed glyco‐molecules in lysosomes. Diagnosis of the particular type of LSD requires identification of the accumulated species, usually from urine. LSD diagnosis has been a target for many years, including use of GC/MS, TLC, NMR, HPLC, FACE and other techniques. Faid et al. take a simple approach that is quite successful and much less time consuming. After permethylating crude urine, a sample is first run on GC/MS to look for free sialic acid, an indicator of one set of diseases, then a cleaned up sample (C_1–8) is analyzed by MALDI/TOF to characterize glycoproteins and glycopeptides present. The glycans were further analyzed by sequential exoglucosidase digestion on the MALDI target. Sulfated glycocompound analysis requires a desulfation step prior to permethylation. Faid, V. et al., Proteomics Clin. Appl. 2008, 2, 528–542. Popular prostate problem receives piercing look Prostate cancer (PCa) is one of the most frequent male cancers. It is also one of the most frequently misdiagnosed cancers. Prostate specific antigen (PSA) has improved the accuracy somewhat but still generates 700 000 false positives requiring biopsies per year in the US. And very low PSA levels don’t assure freedom from PCa. In short, there is no good biomarker for PCa. Theodorescu et al. recognized that anything recovered from blood had a good chance of being at least partially degraded so they looked at first void urine as a more likely source of stable material. Applying capillary electrophoresis coupled to TOF‐MS, the authors developed an “informative” panel of eight peptides that distinguished between first void and mid‐stream samples and a PCa‐specific panel of 12 proteins that detected the disease. Incorporating the 12 proteins with age and free PSA, sensitivity was 91%, specificity 69%. Theodorescu, D. et al., Proteomics Clin. Appl. 2008, 2, 556–570. No candy in candidiasis Candida albicans is a unicellular yeast that is the third most common nosocomial agent in ICU’s, costing ～US$ 40 000 per incident. Early diagnosis of systemic candidiasis (SC) is crucial for successful control of the infection by antifungal therapies. Blood cultures are not fast and tissue biopsies are not sensitive but are the current “gold standards.” Pitarch et al. report here a serum protein marker for SC: anti‐Candida enolase (Eno1p) IgG. Comparing healthy with SC patients for antibodies against Candida cytoplasmic antigens, 15 antigens were recognized by the SC patients. The strongest response was to Eno1p which was the only antigen to produce a response in all 12 patients. The IgG was tested for its suitability as an antigen in Western and capture ELISA tests. Although the tests appear reliable for detecting SC, they were not good for prognosis. A considerable amount of work remains for full qualification of the test. Pitarch, A. et al., Proteomics Clin. Appl. 2008, 2, 596–618.     

Screening of serum samples from Wegener's granulomatosis patients using antibody microarrays

Wegener's Granulomatosis (WG) is an idiopathic granulomatosis autoimmune vasculitis that primarily affects small vessels and is associated with glomerulonephritis and pulmonary granulomatous vasculitis. Anti‐neutrophil cytoplasmic auto‐antibodies (cANCA) against proteinase‐3 are used to identify WG, but ANCA titers are not present in some patients with the localized disease. The objective of this study was to develop an antibody array to help identify protein expression patterns in serum from patients with WG as compared to normals. The arrays were tested for limits of detection, background, and cross reactivity using standard proteins. The arrays were hybridized with either normal patient serum (n = 30) or with serum samples from a population of WG patients (n = 26) that were age and sex matched. Data analysis and curve fitting of the standard dilution series calculated r² values and determined a sensitivity of <50 pg/mL for the majority of proteins. A total of 24 proteins were assessed. Several statistically significant increases (p<0.05) were seen in the expression of: angiotensin converting enzyme‐I, IFN‐γ, IL‐8, s‐ICAM‐1 and s‐VCAM in WG patients as compared to controls. Utilizing the antibody microarray technology has led to the identification of potential biomarkers of vascular injury in the serum of WG patients.     

In pursuit of B-cell synovial autoantigens in rheumatoid arthritis: Confirmation of citrullinated fibrinogen, detection of vimentin, and introducing carbonic anhydrase as a possible new synovial autoantigen

We aimed to investigate potential synovial autoantigens in rheumatoid arthritis (RA) that could trigger the induction of B‐cell autoantibodies. Total protein extract of synovial tissue obtained from seven RA patients was pooled and separated by 1‐DE and 2‐DE. The corresponding blots were probed with sera from RA (n = 30) and disease control samples (n = 30). Protein spots showing a sensitivity of >15% were identified by MS. 1‐D immunoblots revealed one protein band with a specificity in RA of 100%, a sensitivity of 43%, which was identified as fibrinogen β chain. 2‐D analysis revealed the subunits of fibrinogen, especially the β and γ chain, as the most prominent synovial autoantigens. We also identified vimentin, the Sa‐antigen and carbonic anhydrase I as a potentially new synovial autoantigen. The protein patterns of these immunoreactive spots were observed as trains. The spots showing the highest autoimmune reactivity occurred at the acidic side of these trains and were recognized by anticitrullinated protein/peptide antibodies positive RA sera. Antimodified citrulline staining of these patterns confirmed protein citrullination. Therefore, PTMs such as citrullination due to alterations of peptidylarginine deiminase activity or generation of RA‐specific epitopes, should be considered as a trigger in tolerance break.     

Identification of anti-α-enolase autoantibody as a novel serum marker for endometriosis

Diagnosis of endometriosis needs invasive maneuvers. New serum marker that possesses both high sensitivity and high specificity has long been desired. To establish novel serum marker for endometriosis, serum autoantibodies (autoAbs) were investigated using proteomic approach. AutoAbs in sera of endometriotic patients and healthy controls were analyzed using a mesothelial cell line, 2-DE and Western blotting. Proteins in reacted spots were identified using MALDI TOF-MS with MASCOT analysis. ELISAs were established using recombinant proteins and autoAb-titers were estimated in sera of endometriotic patients, disease and healthy controls. Several autoAbs were identified. Anti-α-enolase (Eno1)-autoAb levels in endometriotic patients were significantly elevated compared with both healthy and disease controls. Sensitivity and specificity of serum anti-Eno1-autoAb was nearly comparable to serum CA125. When anti-Eno1-autoAb and CA125 assays were combined, diagnostic sensitivity and accuracy improved. Serum anti-Eno1-autoAb can be a new serum endometriotic marker and it is useful as a supplement assay for CA125. This study validates further clinical evaluation of this novel marker.     

兔抗原生物敏电极的研究

采用石墨电极作为基础电极 ,将待测兔抗原用丝素蛋白溶液固定在基础电极表面 ,根据抗原抗体的单一性识别原理 ,选用山羊抗兔IgG HRP抗体与其选择性结合制作而成 .利用H2 O2 将该生物敏传感器的电位响应信号放大 ,采用直接电位法检测兔抗原的浓度 .结果符合奈斯特响应 ,兔抗原的最低检测浓度 1.0× 10 -10 mol/L ,线性范围 1.0× 10 -8～ 1.0× 10 -10 mol/L ,响应时间为 15s .这种以固定化抗原结合酶标抗体量的多少作为检测抗原浓度的新型酶免疫电极 ,在临床检测、生物医学研究等领域中有着广阔的应用前景     

Proteomics-based identification of HSP60 as a tumor-associated antigen in colorectal cancer

Patients with cancer frequently develop autoantibodies. The identification of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. In this study, we used serological proteomics analysis (SERPA) to identify tumor proteins that elicit humoral response in colorectal cancer (CRC). The CRC cell line HCT116 was used as a source of proteins for 2-DE and subsequent Western blot analysis in which individual serum from patients with CRC was analyzed for autoantibodies. An autoantibody against HSP60 identified by MS was detected in 13 out of 25 patients with CRC and 1 out of 15 healthy subjects. In addition, the HSP60 expressions in tumor tissues collected from 40 patients with CRC were assessed by immunohistochemistry, and serum specimens from 100 patients with cancer and 30 healthy controls were screened for antibody titer to HSP60 by ELISA. The results showed that expressions of HSP60 in tumor tissue and serum antibody titer to HSP60 were significantly higher in patients with CRC than in healthy subjects. Thus, we conclude that the SERPA is an excellent assay for the identification of tumor-associated antigens and tumor markers. The detection of HSP60 may have clinical utility in CRC screening, diagnosis, and immunotherapy.     

Proteomic analysis of sera of asymptomatic,early‐stage patients with Wilson's disease

Wilson's disease (WD) is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues, leading to significant oxidative stress and tissue damage. To date, several diagnostic biomarkers for WD such as serum ceruloplasmin, serum or urine copper levels and copper content in liver have been identified. However, these biomarkers may not be convincing for the diagnosis in some WD patients. To identify additional novel diagnostic biomarkers, we compared the serum protein profiles of asymptomatic childhood WD patients (n=20), without neurologic manifestation or liver cirrhosis, with normal controls (n=13). Fourteen spots, five up‐regulated and nine down‐regulated (>2‐fold), were differentially expressed in WD patients in comparison to normal control on 2‐DE. Among them, three spots were down‐regulated in both male and female WD. MS/MS analysis revealed that the three spots were complement component C3, complement factor B and alpha‐2 macroglobulin. By comparative proteome analysis, complement component C3, complement factor B and alpha‐2 macroglobulin, which are related to oxidative stress and inflammation, turned out to be good candidates for novel diagnostic biomarkers for early stages of WD.     

Thymosin β4 is differentially expressed in the cerebrospinal fluid of Creutzfeldt‐Jakob disease patients: a MALDI‐TOF MS protein profiling study

MALDI‐TOF protein profiling analysis permits the detection of peptides and small proteins in complex protein mixtures with great accuracy. We applied this analysis to cerebrospinal fluid (CSF) from 15 patients affected by Creutzfeldt‐Jakob disease (CJD). We compared the levels of the normalized ion signals of 11 sporadic and 4 genetic CJD forms with those from ten healthy control subjects and eight non‐CJD relapsing‐remitting multiple sclerosis patients. In so doing, we detected 61 differentially expressed ion signals in CJD samples compared to controls. Among the 61 signals, 3 signals had significantly increased levels with high statistical significance (p <0.0001) and were located at 3238.3 m/z, 4963.7 m/z, and 8565.3 m/z. We characterized the 5.0 and 8.6 kDa proteins as thymosin β4 N‐acetylated and free ubiquitin, respectively, while the 3.2‐kDa peptide remained uncharacterized. Although elevated ubiquitin levels have previously been described in CJD, we have demonstrated for the first time the involvement of thymosin β4 in a neurodegenerative disease. To support the validity of thymosin β4 levels obtained by MALDI‐TOF analysis, an independent enzyme immunoassay analysis was performed. Moreover, a validation cohort consisting of CSF from three CJD patients, five healthy subjects, and six non‐CJD relapsing‐remitting multiple sclerosis patients was analyzed in a similar way, yielding superimposable results. We propose that thymosin β4 is a potential new candidate marker for the ante mortem diagnosis of CJD disease.     

Evaluation of a type 1 diabetes serum cohort by SELDI-TOF MS protein profiling

Proteomics analysis of serum from patients with type 1 diabetes (T1D) may lead to novel biomarkers for prediction of disease and for patient monitoring. However, the serum proteome is highly sensitive to sample processing and before proteomics biomarker research serum cohorts should preferably be examined for potential bias between sample groups. SELDI‐TOF MS protein profiling was used for preliminary evaluation of a biological‐bank with 766 serum samples from 270 patients with T1D, collected at 18 different paediatric centers representing 15 countries in Europe and Japan over 2 years (2000–2002). Samples collected 1 (n = 270), 6 (n = 248), and 12 (n = 248) months after T1D diagnosis were grouped across centers and compared. The serum protein profiles varied with collection site and day of analysis; however, markers of sample processing were not systematically different between samples collected at different times after diagnosis. Three members of the apolipoprotein family increased with time in patient serum collected 1, 6, and 12 months after diagnosis (ANOVA, p<0.001). These results support the use of this serum cohort for further proteomic studies and illustrate the potential of high‐throughput MALDI/SELDI‐TOF MS protein profiling for evaluation of serum cohorts before proteomics biomarker research.     

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre‐clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI‐TOF‐MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre‐albumin) and C3a‐desArg by MS/MS and were further validated by antibody‐based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut‐off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a‐desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.     

Investigation of an albumin‐enriched fraction of human serum and its albuminome

The removal of albumin and other high abundance proteins is a routine first step in the analysis of serum and plasma proteomes. However, as albumin can bind proteins and peptides, there is a universal concern as to how the serum proteome is changed by the removal of albumin. To address this concern, the current study was designed to identify proteins and peptides removed from the serum during albumin depletion; to determine which of these are bound to albumin (rather than copurified) and whether the bound proteins are intact proteins or peptide fragments. Sequential, independent analyses including both anti‐albumin antibody (anti‐HSA) affinity chromatography and SEC were used to isolate albumin‐bound proteins. RP‐HPLC and 1‐D SDS‐PAGE were then used to further separate the proteins prior to identification by MS/MS. Finally, whole protein molecular weight (MW) MS measurements coupled with protein coverage obtained by MS were combined to assess whether the bound proteins were intact or peptide fragments. Combining the results from multiple approaches, 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.     

Shotgun immunoproteomics to identify disease‐associated bacterial antigens: Application to human colon cancer

Circulating antibodies reflect a mirror view of invading antigens that are related to infection and cancer. This was recently exemplified by using serum antibodies to capture Streptococcus bovis antigens followed by MS to generate antigen profiles that were diagnostic for colon cancer. These bacterial antigen profiles have a high potential to aid in the immuno‐diagnosis of this disease, as the magnitude of the immune response to bacterial antigens is, in general, superior to the immune response against tumor (self) antigens. In this study, the identity of individual colon cancer‐associated streptococcal antigens was revealed by enrichment of these “diagnostic” antigens by selected patient antibodies followed by high‐accuracy nanoLC‐MS/MS peptide identification. This showed that both the histone‐like protein HlpA and the ribosomal protein Rp L7/L12 are members of the colon cancer‐associated S. bovis immunome. Both antigens also seem to belong to the group of anchorless surface proteins, like 14 additional proteins that were co‐identified in S. bovis cell wall extracts. Among these were the known streptococcal anchorless surface proteins GAPDH and Enolase. Taken together, these data show that shotgun immunoproteomics, combining immunocapture in‐line with LC MS/MS, is a convenient approach for the rapid identification of disease‐associated bacterial antigens.     

Two‐dimensional gel electrophoresis of apolipoprotein C‐III and other serum glycoproteins for the combined screening of human congenital disorders of O‐ and N‐glycosylation

Congenital disorders of glycosylation (CDG) are inherited diseases that can affect not only the N‐glycan (e.g. CDG type I and II) but also the O‐glycan biosynthesis pathway. In the absence of specific clinical symptoms, there is a need for a reliable biological screening of these two groups of CDG. Using a few microlitres of human serum, 2‐DE and immunoblotting were applied to the separation and simultaneous detection of the isoforms of the O‐glycosylated protein apolipoprotein C‐III (apoC‐III) and of four N‐glycosylated proteins, namely alpha‐antitrypsin, alpha‐1 acid glycoprotein, haptoglobin and transferrin. For the study of O‐glycosylation, this technique allowed the reliable separation of the three fractions of apoC‐III and the determination of normal percentage values in an adult population. Concerning N‐glycosylation, the study of serum samples from patients with CDG type Ia revealed marked abnormalities systematically affecting the four 2‐DE separated N‐linked glycoproteins. 2‐DE coupled to immunoblotting using a mixture of specific antibodies could be easily and reliably employed for the combined screening of both N‐ and O‐glycosylation disorders in humans.     

Apolipoprotein A1: A new serum marker correlated to JAK2 V617F proportion at diagnosis in patients with polycythemia vera

Polycythemia vera (PV) is a myeloproliferative disorder (MPD) characterized by an acquired gain-of-function mutation of the JAK2 protein (JAK2 V617F). Allele-specific quantitative PCR has showed a JAK2 V617F dosage effect on haematological and clinical parameters of PV at diagnosis, but it is unknown whether the level of certain serum proteins might correlate with the proportion of mutated JAK2. Taking into account that such proteins could represent useful prognostic marker, we investigated the serum protein profile of PV patients by SELDI-TOF MS. We identified apolipoprotein A1 (Apo-A1) as a serum marker correlated to the percentage of JAK2 V617F alleles; Apo-A1 expression being the highest for PV patients with more than 75% of mutated alleles. Immuno-assay on an automated random immuno-analyser confirmed the correlation between Apo-A1 concentrations and JAK2 V617F percentages, and showed that serum Apo-A1 assay allowed the specific discrimination of PV patients with high levels of mutated alleles (≥75%). These data suggest that Apo-A1 assay could be a useful assay for the stratification of PV patients at diagnosis.     

Comparative proteomic analysis of differentially expressed proteins in an in vitro cellular carcinogenesis model of oral squamous cell carcinoma

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2‐DE and LC‐tandem mass chromatography to separate and identify differentially expressed proteins. Forty‐five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.     

Dynamic anti‐windup design in anticipation of actuator saturation

In a traditional anti‐windup design, the anti‐windup mechanism is set to be activated as soon as the control signal saturates the actuator. A recent innovation of delaying the activation of the anti‐windup mechanism, both static and dynamic, until the saturation reaches a certain level of severity has led to a performance improvement of the resulting closed‐loop system. It has been shown that significant further performance improvements can be obtained by activating a static anti‐windup mechanism in anticipation of actuator saturation, instead of immediate or delayed activation. This paper demonstrates that anticipatory activation of a dynamic anti‐windup mechanism would also lead to significant performance improvements over both the immediate and delayed activation schemes.Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of Apolipoprotein AI as a serum biomarker of chronic kidney disease in liver transplant recipients, using proteomic techniques

Chronic kidney disease (CKD) is a common complication post‐orthotopic liver transplantation (OLT). Development of CKD is detected by monitoring serum urea and creatinine, however disease can occasionally be at an advanced stage before they become abnormal. Therefore, more accurate parameters are required. In order to identify novel biomarkers of CKD, serum was obtained from 47 OLT recipients with CKD (glomerular filtration rate <60 mL/min) and 23 with normal renal function (glomerular filtration rate >90 mL/min). Using the proteomic technique SELDI‐TOF‐MS, three protein biomarkers (55.6 kDa, 9.5 kDa and 11.4 kDa) were identified that, together, could stratify patients into cases or controls with a sensitivity and specificity of 93.6 and 91.3%, respectively. The area under the curve was 0.94. The primary splitter of the groups at 55.6 kDa was an alternative version of a molecule at 27.8 kDa, which was subsequently identified by 1‐D SDS‐PAGE and LC‐ESI‐MS/MS to be Apolipoprotein AI. Protein expression was shown to be reduced in CKD, by both ELISA (p = 0.057) and Western blot analysis (p = 0.003). Apolipoprotein AI is a novel, accurate marker of CKD post‐OLT. It does require further validation in a large, more diverse patient population but could potentially improve detection of CKD.