Ekaterina Mirgorodskaya

Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry

A simple reversed-phase nano-column purification and sample preparation technique is described, which markedly improves the mass spectrometric analysis of complex and contaminated peptide mixtures by matrix-assisted laser desorption/ionization (MALDI). The method is simple, fast and utilizes only low-cost disposables. After loading the sample on the column and a subsequent washing step, the analyte molecules are eluted with 50-100 nl of matrix solution directly on to the MALDI/MS target. The washing step ensures removal of a wide range of contaminants. The small bed volume of the column allows efficient sample concentration and the elution process yields very small sample spots. This simplifies the analysis and minimizes discrimination effects due to sample heterogeneity, because the desorption/ionization laser simultaneously irradiates a large portion of the sample. Taken together, these features of the method significantly improve the sensitivity for MALDI/MS analysis of contaminated peptide samples compared with the commonly used sample preparation procedures. This is demonstrated with in-gel tryptic digests of proteins from human brain that were separated by 2D gel electrophoresis. Furthermore, it is shown that with this method 2,5-dihydroxybenzoic acid (DHB) acts as an efficient matrix for peptide mapping. Both detection sensitivity and sequence coverage are comparable to those obtained with the currently preferred matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The higher stability of peptide ions generated with DHB compared with CHCA is advantageous when analyzing fragile sample molecules. Therefore, the method described here is also of interest for the use of Fourier transform ion cyclotron resonance (FT-ICR) or ion-trap mass analyzers.

Immunohistochemical Study of the Expression of MUC6 Mucin and Co-expression of Other Secreted Mucins (MUC5AC and MUC2) in Human Gastric Carcinomas

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.

Development and characterization of an antibody directed to an alpha-N-acetyl-D-galactosamine glycosylated MUC2 peptide.

In an attempt to raise anti-Tn antibodies, an α-N-acetyl-D-galactosamine glycosylated peptide based on the tandem repeat of the intestinal mucin MUC2 was used as an immunogen. The MUC2 peptide (PTTTPISTTTMVTPTPTPTC) was glycosylated in vitro using concentrated α-N-acetylgalactosaminyltransferases activity from porcine submaxillary glands which resulted in the incorporation of 8–9 mol of Ga/NAc. Rabbits and mice developed specific anti-MUC2-GalNAc glycopeptide antibodies and no detectable anti-Tn antibodies. Anti-glycopeptide antibodies did not show reactivity with the unglycosylated MUC2 peptide or with other GalNAc glycosylated peptides. A mouse monoclonal antibody (PMH1) representative of the observed immune response was generated and its immunohistological reactivity analysed in normal tissues. PMH1 reacted similarly to other anti-MUC2 peptide antibodies. However, in some cells the staining was not restricted to the supranuclear area but extended to the entire cytoplasm. In addition, PMH1 reacted with purified colonic mucin by Western blot analysis suggesting that PMH1 reacted with some glycoforms of MUC2. The present work presents a useful approach for development of anti-mucin antibodies directed to different glycoforms of individual mucins.

Peptidome Analysis of Cerebrospinal Fluid by LC-MALDI MS

We report on the analysis of endogenous peptides in cerebrospinal fluid (CSF) by mass spectrometry. A method was developed for preparation of peptide extracts from CSF. Analysis of the extracts by offline LC-MALDI MS resulted in the detection of 3,000–4,000 peptide-like features. Out of these, 730 peptides were identified by MS/MS. The majority of these peptides have not been previously reported in CSF. The identified peptides were found to originate from 104 proteins, of which several have been reported to be involved in different disorders of the central nervous system. These results support the notion that CSF peptidomics may be viable complement to proteomics in the search of biomarkers of CNS disorders.

Exhaled Endogenous Particles Contain Lung Proteins

BACKGROUND We recently developed a novel, noninvasive method for sampling nonvolatile material from the distal airways. The method is based on the collection of endogenous particles in exhaled air (PEx). The aim of this study was to characterize the protein composition of PEx and to verify that the origin of PEx is respiratory tract lining fluid (RTLF). METHOD Healthy individuals exhaled into the sampling device, which collected PEx onto a silicon plate inside a 3-stage impactor. After their extraction from the plates, PEx proteins were separated by SDS-PAGE and then analyzed by LC-MS. Proteins were identified by searching the International Protein Index human database with the Mascot search engine. RESULTS Analysis of the pooled samples identified 124 proteins. A comparison of the identified PEx proteins with published bronchoalveolar lavage (BAL) proteomic data showed a high degree of overlap, with 103 (83%) of the PEx proteins having previously been detected in BAL. The relative abundances of the proteins were estimated according to the Mascot exponentially modified protein abundance index protocol and were in agreement with the expected protein composition of RTLF. No amylase was detected, indicating the absence of saliva protein contamination with our sampling technique. CONCLUSIONS Our data strongly support that PEx originate from RTLF and reflect the composition of undiluted RTLF.

Surfactant protein A and albumin in particles in exhaled air

In this study we test the hypothesis that endogenous particles in exhaled air (PEx), non-invasively sampled from lower airways, are well suited for the analysis of respiratory tract lining fluid (RTLF) proteins, i.e., surfactant protein A (SP-A) and albumin. Ten healthy volunteers were included in the study and participated in two sampling sessions. Blood, exhaled breath condensate (EBC) and PEx were collected at each session. 100 L of breath were collected for each exhaled sample. Serum and exhaled samples were analyzed for SP-A using an in-house ELISA. Albumin was analyzed in exhaled samples using a commercial ELISA kit. SP-A detection rates were 100%, 21%, and 89% for PEx, EBC and serum, respectively. Albumin was detected in PEx, but not in EBC. SP-A measurements in PEx showed good repeatability with an intra-individual coefficient of variation of 13%. Both SP-A and albumin showed significant correlation to mass of PEx (r(s) = 0.93, p < 0.001 and r(s) = 0.86, p = 0.003, respectively). Sampling and analysis of PEx is a valid non-invasive method to monitor RTLF proteins sampled from the lower respiratory tract, as demonstrated here by example of SP-A and albumin analysis.

Potential of Mass Spectrometry in Developing Clinical Laboratory Biomarkers of Nonvolatiles in Exhaled Breath

BACKGROUND Exhaled breath contains nonvolatile substances that are part of aerosol particles of submicrometer size. These particles are formed and exhaled as a result of normal breathing and contain material from distal airways of the respiratory system. Exhaled breath can be used to monitor biomarkers of both endogenous and exogenous origin and constitutes an attractive specimen for medical investigations. CONTENT This review summarizes the present status regarding potential biomarkers of nonvolatile compounds in exhaled breath. The field of exhaled breath condensate is briefly reviewed, together with more recent work on more selective collection procedures for exhaled particles. The relation of these particles to the surfactant in the terminal parts of the respiratory system is described. The literature on potential endogenous low molecular weight compounds as well as protein biomarkers is reviewed. The possibility to measure exposure to therapeutic and abused drugs is demonstrated. Finally, the potential future role and importance of mass spectrometry is discussed. SUMMARY Nonvolatile compounds exit the lung as aerosol particles that can be sampled easily and selectively. The clinical applications of potential biomarkers in exhaled breath comprise diagnosis of disease, monitoring of disease progress, monitoring of drug therapy, and toxicological investigations.

Surfactant Protein A in Exhaled Endogenous Particles Is Decreased in Chronic Obstructive Pulmonary Disease (COPD) Patients: A Pilot Study

Background Exhaled, endogenous particles are formed from the epithelial lining fluid in small airways, where surfactant protein A (SP-A) plays an important role in pulmonary host defense. Based on the knowledge that chronic obstructive pulmonary disease (COPD) starts in the small airway epithelium, we hypothesized that chronic inflammation modulates peripheral exhaled particle SP-A and albumin levels. The main objective of this explorative study was to compare the SP-A and albumin contents in exhaled particles from patients with COPD and healthy subjects and to determine exhaled particle number concentrations. Methods Patients with stable COPD ranging from moderate to very severe (n = 13), and healthy non-smoking subjects (n = 12) were studied. Subjects performed repeated breath maneuvers allowing for airway closure and re-opening, and exhaled particles were optically counted and collected on a membrane using the novel PExA® instrument setup. Immunoassays were used to quantify SP-A and albumin. Results COPD patients exhibited significantly lower SP-A mass content of the exhaled particles (2.7 vs. 3.9 weight percent, p = 0.036) and lower particle number concentration (p<0.0001) than healthy subjects. Albumin mass contents were similar for both groups. Conclusions Decreased levels of SP-A may lead to impaired host defense functions of surfactant in the airways, contributing to increased susceptibility to COPD exacerbations. SP-A in exhaled particles from small airways may represent a promising non-invasive biomarker of disease in COPD patients.

Mass spectrometric identification of a stable catalytic cysteinesulfinic acid residue in an enzymatically active chemically modified glucoamylase mutant

Mass spectrometric identification of cysteinsulfinic acid resulting in restoration of activity of chemically modified Glu400 Cys catalytic-base glucoamylase (GA) mutants is described. This oxidation unexpectedly occurred during attempts to carboxyalkylate the Cys400 GA mutant using three different alkylation reagents. However, mass spectrometric peptide mapping did not show the presence of carboxyalkylation of the Cys400 residue but suggested an oxidation to cysteinsulfinic acid based on the observed mass increment. The presence of cysteinsulfinic acid was confirmed by employing matrix-assisted laser desorption/ionization mass spectrometry combined with post-source decay analysis. Furthermore, strong enhancement of metastable fragmentation was observed for peptides containing oxidized Cys in comparison with non-oxidized peptide.

The effect of exhalation flow on endogenous particle emission and phospholipid composition

Exhaled particles constitute a micro-sample of respiratory tract lining fluid. Inhalations from low lung volumes generate particles in small airways by the airway re-opening mechanism. Forced exhalations are assumed to generate particles in central airways by mechanisms associated with high air velocities. To increase knowledge on how and where particles are formed, different breathing manoeuvres were compared in 11 healthy volunteers. Particles in the 0.41-4.55μm diameter range were characterised and sampled. The surfactant lipid dipalmitoylphosphatidylcholine (DPPC) was quantified by mass spectrometry. The mass of exhaled particles increased by 150% (95% CI 10-470) for the forced exhalation and by 470% (95% CI 150-1190) for the airway re-opening manoeuvre, compared to slow exhalations. DPPC weight percent concentration (wt%) in particles was 2.8wt% (95%CI 1.4-4.2) and 9.4wt% (95%CI 8.0-10.8) for the forced and the airway re-opening manoeuvres, respectively. In conclusion, forced exhalation and airway re-opening manoeuvres generate particles from different airway regions having different DPPC concentration.