Towards stable diagnostic setups in clinical proteomics: Absolute quantitation of peptide biomarkers using MALDI‐TOF‐MS

In routine clinical diagnostics, peptide biomarkers are most commonly quantified using immunological techniques but these methods often lack sensitivity and/or specificity. Hence, quantitative mass spectrometry detection is desirable as an alternative diagnostic tool. To date, quantitative mass spectrometry is mostly based on ESI‐MS coupled to LC, requiring highly sophisticated instrumentation and knowledge and is time consuming and expensive. In contrast, MALDI‐TOF‐MS is a very simple, sensitive and rapid method for the detection of peptide biomarkers. However, the infeasibility of absolute quantification has been a tremendous handicap to the use of MS in stable clinical diagnostics. Here, we describe the development of a technical platform based on ClinProt particles and heavy‐isotope internal peptide standards for the fast and reliable preparation of samples. This combines the advantages of MALDI‐TOF as a read‐out system with absolute quantitation of peptide biomarkers. As a proof‐of‐concept, this platform was successfully employed for the absolute determination of the concentration of the highly abundant serum peptide des‐Ala‐Fibrinopeptide A in 45 serum samples from healthy donors. Such technology essentially contributes to the development of a stable MALDI‐TOF‐MS‐based clinical assay.     

Two dimensional gel electrophoresis of apolipoprotein C‐III and MALDI‐TOF MS are complementary techniques for the study of combined defects in N‐ and mucin type O‐glycan biosynthesis

In the field of diseases related to glycosylation disorders, congenital defects associated with abnormalities in both O‐ and N‐glycosylation of proteins constitute arising novel entities. Defects in subunits of the conserved oligomeric Golgi protein complex have been shown to be involved in an important part of previously unsolved CDG type II combining abnormalities in both mucin type core1 O‐ and N‐glycans; furthermore, recent studies revealed that autosomal recessive cutis laxa type II could also be associated with such combined glycosylation defects. Based on the studies of serum samples from three patients including a case of cutis laxa, we present here evidence that 2‐DE of apolipoprotein C‐III in combination with MALDI‐TOF‐MS analysis of serum O‐ and N‐glycans allow the detection and the biochemical characterization of these newly recognized glycosylation disorders.     

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.     

Protein content in aqueous humor from patients with pseudoexfoliation (PEX) investigated by capillary LC MALDI‐TOF/TOF MS

Analysis of proteins in human body fluids is challenging since the composition of the sample often is rather complex. Here we present a method for analysis of proteins in aqueous humor from two groups of cataract patients, with and without pseudoexfoliation (PEX). Aqueous humor is an extracellular fluid contained in the anterior chamber of the eye between the cornea and iris. The limited volume of sample requires sophisticated analysis techniques. Our method is based on a total tryptic digestion of the sample followed by capillary LC‐MALDI MS and MS/MS analysis of the peptides. The method is rapid, efficient and suitable as a complement or alternative to more commonly used methods based on gel electrophoretic experiments. With this method we found and unambiguously identified 30 nonredundant proteins. Proteins found include general transport proteins such as albumin and apolipoprotein A1 but also specific proteins involved in immune response, such as complement factors. Cystatin C, clusterin, and crystallins were also found. Although the number of proteins was roughly the same in both groups there was a significant difference in their identities. These findings may give some new insights into the pathophysiology of the PEX syndrome.     

Gaussian mixture decomposition in the analysis of MALDI‐TOF spectra

The article presents a method of protein matrix‐assisted laser desorption and ionization‐time of flight (TOF) spectra analysis. The method performs peaks detection. Spectra are analysed with Gaussian mixture decomposition. The results obtained are used for peaks identification purposes. The concept of the method is that a single peak is represented by one Gaussian distribution. The expectation‐maximization algorithm and maximum likelihood rule are used for spectra processing. The analysis can be done for a set of spectra with use of the mean spectrum, or it may be performed for a single spectrum at a time. The number of mixture‐model components is estimated by the Bayesian information criterion. Before the main analysis, a few pre‐processing steps need to be done. Spectra should be subjected to calibration, normalization, denoising, baseline correction, etc. The aim of the work is to identify peptides in the analysed sample on the basis of the parameters of the mixture model and to find differences between spectra in the analysed set.     

Design of a 10 W high‐efficiency balanced power amplifier using a combination of load‐pull and load‐line methods for ISM band

In this article, a 10 W power amplifier has been designed and constructed at 2.4 GHz. The source and load‐pull impedance data published by the manufacturer at a nearby frequency of 2.5 GHz have been adopted to power match the transistor at the intended design frequency. For this purpose, the linear model of the GaN transistor has been derived from the S‐parameter data. The load‐line at the dependent current source plane and the impedance at the intrinsic gate‐source capacitance have been simulated in the presence of the source and load‐pull impedances at 2.5 GHz. The extracted impedances have been retained in the design of the power amplifier at 2.4 GHz. In a novel approach, the input and output matching circuits interacted with the linear model of the transistor to provide the same load‐line conditions at the virtual drain plane and the intrinsic gate‐source capacitance plane. In contrast to conventional load‐pull methods that give no information about the harmonic terminations, harmonic terminations can be easily controlled in this method. The insight into the transistor linear model allows the harmonic terminations at the virtual drain plane to be set to low values for proper class‐B operation.     

Targeted detection of prostate cancer proteins in serum using heavy‐isotope‐labeled‐peptide standards and MALDI‐TOF/TOF

Proteins released from cancer tissues to patient sera can potentially be used to achieve sensitive, specific, and early detection of cancer by means of blood tests. In this study, we used a platform that combines glycopeptide capture, heavy‐isotope‐labeled‐peptide standards, and liquid chromatography coupled to tandem mass spectrometry to determine which glycoproteins from prostate cancer can be detected in sera from patients with early‐stage prostate cancer. The detection limit for prostate‐specific antigen in serum was 3.44 ng/mL; thus, direct identification of low abundance, cancer‐specific proteins was achieved using our platform. We showed that prostatic acid phosphatase and membrane metallo‐endopeptidase that were detected in sera were preferentially expressed in prostate cancer tissues. Levels of these two proteins were elevated in biopsy‐positive patients but not biopsy‐negative groups. Therefore, these two proteins are candidate biomarkers for analysis of patient samples with levels of prostate‐specific antigen in the diagnostic gray zone.