Advances in MALDI mass spectrometry in clinical diagnostic applications.

The concept of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was first reported in 1985. Since then, MALDI MS technologies have been evolving, and successfully used in genome, proteome, metabolome, and clinical diagnostic research. These technologies are high-throughput and sensitive. Emerging evidence has shown that they are not only useful in qualitative and quantitative analyses of proteins, but also of other types of biomolecules, such as DNA, glycans, and metabolites. Recently, parallel fragmentation monitoring (PFM), which is a method comparable to selected reaction monitoring, has been reported. This highlights the potentials of MALDI-TOF/TOF tandem MS in quantification of metabolites. Here we critically review the applications of the major MALDI MS technologies, including MALDI-TOF MS, MALDI-TOF/TOF MS, SALDI-TOF MS, MALDI-QqQ MS, and SELDI-TOF MS, to the discovery and quantification of disease biomarkers in biological specimens, especially those in plasma/serum specimens. Using SELDI-TOF MS as an example, the presence of systemic bias in biomarker discovery studies employing MALDI-TOF MS and its possible solutions are also discussed in this chapter. The concepts of MALDI, SALDI, SELDI, and PFM are complementary to each other. Theoretically, all these technologies can be combined, leading to the next generation of the MALDI MS technologies. Real applications of MALDI MS technologies in clinical diagnostics should be forthcoming.

Gut-associated biomarkers L-FABP, I-FABP, and TFF3 and LIT score for diagnosis of surgical necrotizing enterocolitis in preterm infants.

OBJECTIVES: To evaluate the use of gut barrier proteins, liver-fatty acid binding protein (L-FABP), intestinal-fatty acid binding protein (I-FABP), and trefoil factor 3 (TFF3), as biomarkers for differentiating necrotizing enterocolitis (NEC) from septicemic/control infants and to identify the most severely affected surgical NEC from nonsurgical NEC infants. BACKGROUND: Clinical features and routine radiologic investigations have low diagnostic utilities in identifying surgical NEC patients. METHODS: The diagnostic utilities of individual biomarkers and the combination of biomarkers, the LIT score, were assessed among the NEC (n = 20), septicemia (n = 40), and control groups (n = 40) in a case-control study for the identification of proven NEC and surgical NEC infants. RESULTS: Plasma concentrations of all gut barrier biomarkers and the LIT score were significantly higher in the NEC than in the septicemia or control group (P < 0.01). Using median values of biomarkers and the LIT score in the NEC group as cutoff values for identifying NEC from septicemic/control cases, all had specificities of 95% or more and sensitivities of 50%. Significantly higher levels of biomarkers and the LIT score were found in infants with surgical NEC than in nonsurgical NEC cases (P ≤ 0.02). The median LIT score of 4.5 identified surgical NEC cases with sensitivity and specificity of 83% and 100%%, respectively. A high LIT score of 6 identified nonsurvivors of NEC with sensitivity and specificity of 78% and 91%, respectively. CONCLUSIONS: The LIT score can effectively differentiate surgical NEC from nonsurgical NEC infants and nonsurvivors of NEC from survivors at the onset of clinical presentation. Frontline neonatologists and surgeons may, therefore, target NEC infants who are most in need of close monitoring and those who may benefit from early surgical intervention.

Identification and characterization of tropomyosin 3 associated with granulin-epithelin precursor in human hepatocellular carcinoma.

BACKGROUND AND AIM: Granulin-epithelin precursor (GEP) has previously been reported to control cancer growth, invasion, chemo-resistance, and served as novel therapeutic target for cancer treatment. However, the nature and characteristics of GEP interacting partner remain unclear. The present study aims to identify and characterize the novel predominant interacting partner of GEP using co-immunoprecipitation and mass spectrometry. METHODS AND RESULTS: Specific anti-GEP monoclonal antibody was used to capture GEP and its interacting partner from the protein extract of the liver cancer cells Hep3B. The precipitated proteins were analyzed by SDS-PAGE, followed by mass spectrometry and the protein identity was demonstrated to be tropomyosin 3 (TPM3). The interaction has been validated in additional cell models using anti-TPM3 antibody and immunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressions were then examined by real-time quantitative RT-PCR in clinical samples, and their transcript levels were significantly correlated. Elevated TPM3 levels were observed in liver cancer compared with the adjacent non-tumorous liver, and patients with elevated TPM3 levels were shown to have poor recurrence-free survival. Protein expression of GEP and TPM3 was observed only in the cytoplasm of liver cancer cells by immunohistochemical staining. CONCLUSIONS: TPM3 is an interacting partner of GEP and may play an important role in hepatocarcinogenesis.

A proteomic approach in investigating the hepatoprotective mechanism of Schisandrin B: role of raf kinase inhibitor protein.

To identify key proteins involved in the hepatoprotection afforded by schisandrin B (Sch B), we used a proteomic approach to screen proteins that were specifically regulated by Sch B in mouse livers and to investigate the role of the proteins in hepatoprotection. Thirteen proteins were specifically activated or suppressed by Sch B treatment. Among the 13 proteins, Raf kinase inhibitor protein (RKIP) was postulated to be the key regulator involved in the development of hepatotoxin-induced cellular damage. The results indicated that the downregulation of RKIP by antisense RKIP vector transfection led to the activation of the Raf-1/MEK/ERK signaling pathway, as evidenced by increases in the level of MEK/ERK phosphorylation and the level of nuclear factor erythroid 2-related factor 2 in the nucleus. The signaling effect produced by RKIP downregulation resembled that triggered by Sch B, wherein both treatments resulted in a decrease in the extent of carbon tetrachloride-induced apoptotic cell death in AML12 hepatocytes. Overexpression of RKIP by the sense RKIP transfection vector or the inhibition of MEK kinase by PD98059 was able to abrogate the cytoprotective effect of Sch B in the hepatocytes. The results indicate that Sch B triggers the Raf/MEK/ERK signaling pathway, presumably by downregulating RKIP, thereby protecting against carbon tetrachloride-induced cytotoxicity.

A magnetic bead-based serum proteomic fingerprinting method for parallel analytical analysis and micropreparative purification.

ProteinChip surface-enhanced laser desorption/ionization technology and magnetic beads-based ClinProt system are commonly used for semi-quantitative profiling of plasma proteome in biomarker discovery. Unfortunately, the proteins/peptides detected by MS are non-recoverable. To obtain the protein identity of a MS peak, additional time-consuming and material-consuming purification steps have to be done. In this study, we developed a magnetic beads-based proteomic fingerprinting method that allowed semi-quantitative proteomic profiling and micropreparative purification of the profiled proteins in parallel. The use of different chromatographic magnetic beads allowed us to obtain different proteomic profiles, which were comparable to those obtained by the ProteinChip surface-enhanced laser desorption/ionization technology. Our assays were semi-quantitative. The normalized peak intensity was proportional to concentration measured by immunoassay. Both intra-assay and inter-assay coefficients of variation of the normalized peak intensities were in the range of 4-30%. Our method only required 2 microL of serum or plasma for generating enough proteins for semi-quantitative profiling by MALDI-TOF-MS as well as for gel electrophoresis and subsequent protein identification. The protein peaks and corresponding gel spots could be easily matched by comparing their intensities and masses. Because of its high efficiency and reproducibility, our method has great potentials in clinical research, especially in biomarker discovery.

Enhanced detection of early hepatocellular carcinoma by serum SELDI-TOF proteomic signature combined with alpha-fetoprotein marker.

BACKGROUND: Biomarkers for accurate diagnosis of early hepatocellular carcinoma (HCC) are limited in number and clinical validation. We applied SELDI-TOF-MS ProteinChip technology to identify serum profile for distinguishing HCC and liver cirrhosis (LC) and to compare the accuracy of SELDI-TOF-MS profile and alpha-fetoprotein (AFP) level in HCC diagnosis. PATIENTS AND METHODS: Serum samples were obtained from 120 HCC and 120 LC patients for biomarker discovery and validation studies. ProteinChip technology was employed for generating SELDI-TOF proteomic features and analyzing serum proteins/peptides. RESULTS: A diagnostic model was established by CART algorithm, which is based on 5 proteomic peaks with m/z values at 3324, 3994, 4665, 4795, and 5152. In the training set, the CART algorithm could differentiate HCC from LC subjects with a sensitivity and specificity of 98% and 95%, respectively. The results were assessed in blind validation using separate cohorts of 60 HCC and 60 LC patients, with an accuracy of 83% for HCC and 92% for LC patients. The diagnostic odd ratio (DOR) indicated that SELDI-TOF proteomic signature could achieve better diagnostic performance than serum AFP level at a cutoff of 20 ng/mL (AFP(20)) (92.72 vs 9.11), particularly superior for early-stage HCC (87% vs 54%). Importantly, a combined use of both tests could enhance the detection of HCC (sensitivity, 95%; specificity, 98%; DOR, 931). CONCLUSION: Serum SELDI-TOF proteomic signature, alone or in combination with AFP marker, promises to be a good tool for early diagnosis and/screening of HCC in at-risk population with liver cirrhosis.