Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Inhibition of Orientia tsutsugamushi infection by a truncated recombinant 56‐kDa outer membrane protein

Aims: The objective of this study was to evaluate recombinant 56‐kDa outer membrane protein as a potential inhibitor to infection from Orientia tsutsugamushi. Methods and Results: The 56‐kDa protein was cloned and expressed in an Escherichia coli system, and the degree of target cell attachment to immobilized 56‐kDa protein was measured in a cell adhesion assay. The results showed that the 56‐kDa protein has an ability to attach HeLa cells. Furthermore, treatment of target cells with a truncated 56‐kDa 1–418 (amino acid residues) protein inhibited target cell infection by O. tsutsugamushi, demonstrated with an indirect immunofluorescence antibody assay. Conclusions: The truncated 56‐kDa protein (a.a. 1–418) plays an important role in O. tsutsugamushi infection, and the 56‐kDa protein could be useful and effective in the inhibition of O. tsutsugamushi attachment and infection. Significance and Impact of the Study: The attachment of the 56‐kDa protein to target cells was directly determined by in vitro adherence test, and the invasion of target cells by O. tsutsugamushi was inhibited by treating the target cells with a truncated 56‐kDa protein.     

Proteomic profiling of Cronobacter turicensis 3032, a food‐borne opportunistic pathogen

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface‐associated and whole‐cell proteins by two complementary proteomics approaches, 1D‐SDS‐PAGE combined with LC‐ESI‐MS/MS and 2D‐LC‐MALDI‐TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin‐receptor protein for the uptake of siderophore‐bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.     

Differential proteomic analysis of lymphatic,venous, and arterial endothelial cells extracted from bovine mesenteric vessels

Differential protein profiling by 2‐D PAGE is generally useful in biomarker discovery, proteome analysis and routine sample preparation prior to analysis by MS. The goal of this study was to compare 2‐D PAGE‐resolved protein profile of lymphatic endothelial cells to those of venous, and arterial endothelial cells isolated from lymphatic and blood vessels of bovine mesentery (bm). Three 2‐D PAGE electrophoretograms were produced for each of the three cell types and quantitatively analyzed. Protein identification by LC‐MS/MS was performed to identify 39 proteins found to be present at statistically significantly different levels in the three cell types (p<0.05). Most of the 39 proteins have not been previously reported in EC proteomic studies of 2‐D PAGE electrophoretograms. Three proteins, HSPA1B (HSP70 family member), HSPB1 (HSP27 family member), and UBE2D3 (a member of E2 ubiquitin‐conjugating enzymes) found to be at highest levels in bm arterial endothelial cells, bm venous endothelial cells, and bm lymphatic endothelial cells, respectively, were validated by immunoblotting with appropriate antibodies. The lack of substantial overlap between our results and those of other groups' comparative studies are discussed. Functional implications of differences in levels of various proteins identified in the three cell types are also discussed.     

Mass spectrometry in plant proteomic analysis

Abstract

The current revolution in proteomics has been generated by the combination of very sensitive mass spectrometers coupled to microcapillary liquid chromatography, specific proteolysis of protein mixtures and software that is capable of searching vast numbers of mass measurements against predicted peptides from sequenced genomes. The challenges of post‐genomic plant biology include characterization of protein function, post‐translational modifications and composition of protein complexes as well as deciphering protein complements in intracellular compartments – proteomes of cell organelles. In this review we summarize the current mass spectrometry methods currently being used in plant proteomics and discuss the various tagging strategies that are being used for purification and proteomic analysis of plant protein complexes.

Abbreviations: BCCD, biotin carboxyl carrier protein domain; CBP, calmodulin‐binding protein; CID, collision‐induced dissociation; ESI, electrospray ionization; EST, expressed sequence tag; FT‐ICR, Fourier transform ion cyclotron resonance; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin; HILEP, hydroponic isotope labelling of entire plants; His, histidine; HPB, HA–PreScission–Biotin; HPLC, high‐performance liquid chromatography; ICAT, isotope‐coded affinity tags; ICPL, isotope‐coded protein label; iTRAQ, isobaric tag for relative and absolute quantification; LC, liquid chromatography; MALDI, matrix‐assisted laser desorption ionization; MBP, maltose‐binding protein; MS, mass spectrometry; SDS‐PAGE, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis; SILAC, stable isotope labelling with amino acids in cell culture; SILIP, stable isotope labelling in planta; Strep, streptavidin; TAP, tandem affinity purification; TBP, TATA‐box‐binding protein; TOF, time‐of‐flight; UPLC, ultraperformance liquid chromatography     

Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ‐coupled two‐dimensional LC‐MS/MS

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ‐coupled 2D LC‐MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25‐fold at p < 0.05) and 55 proteins were downregulated (<0.8‐fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen‐like (CD5L), hyaluronan‐binding protein 2 (HABP2), and retinol‐binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.     

Mass spectrometric analysis of GABAA receptor subtypes and phosphorylations from mouse hippocampus

The brain GABA_A receptor (GABA_AR) is a key element of signaling and neural transmission in health and disease. Recently, complete sequence analysis of the recombinant GABA_AR has been reported, separation and mass spectrometrical (MS) characterisation from tissue, however, has not been published so far. Hippocampi were homogenised, put on a sucrose gradient 10–69% and the layer from 10 to 20% was used for extraction of membrane proteins by a solution of Triton X‐100, 1.5 M aminocaproic acid in the presence of 0.3 M Bis‐Tris. This mixture was subsequently loaded onto blue native PAGE (BN‐PAGE) with subsequent analysis on denaturing gel systems. Spots from the 3‐DE electrophoretic run were stained with Colloidal Coomassie Brilliant Blue, and spots with an apparent molecular weight between 40 and 60 kDa were picked and in‐gel digested with trypsin, chymotrypsin and subtilisin. The resulting peptides were analysed by nano‐LC‐ESI‐MS/MS (ion trap) and protein identification was carried out using MASCOT searches. In addition, known GABA_AR‐specific MS information taken from own previous studies was used for searches of GABA_AR subunits. β‐1, β‐2 and β‐3, θ and ρ‐1 subunits were detected and six novel phosphorylation sites were observed and verified by phosphatase treatment. The method used herein enables identification of several GABA_AR subunits from mouse hippocampus along with phosphorylations of β‐1 (T227, Y230), β‐2 (Y215, T439) and β‐3 (T282, S406) subunits. The procedure forms the basis for GABA_AR studies at the protein chemical rather than at the immunochemical level in health and disease.     

Proteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer)

Herbivorous insects can cause severe cellular changes to plant foliage following infestations, depending on feeding behaviour. Here, a proteomic study was conducted to investigate the influence of green peach aphid (Myzus persicae Sulzer) as a polyphagous pest on the defence response of Arabidopsis thaliana (L.) Heynh after aphid colony establishment on the host plant (3 days). Analysis of about 574 protein spots on 2‐DE gels revealed 31 differentially expressed protein spots. Twenty out of these 31 differential proteins were selected for analysis by mass spectrometry. In 12 of the 20 analysed spots, we identified seven and nine proteins using MALDI‐TOF‐MS and LC‐ESI‐MS/MS, respectively. Of the analysed spots, 25% contain two proteins. Different metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defence response and translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis by M. persicae aphids. It provides valuable insights into the complex responses of plants to biological stress, particularly for herbivorous insects with sucking feeding behaviour.     

Biochemical characterization and molecular modeling of a unique lipase from Staphylococcus arlettae JPBW‐1

A three‐step purification of a unique lipase with halo‐, solvent‐, detergent‐, and thermo‐tolerance from Staphylococcus arlettae JPBW‐1 gave raise to a 27‐fold purification with a specific activity of 32.5 U/mg. The molecular weight of the purified lipase was estimated to be 45 kDa using SDS–PAGE, and its amino acid sequence was characterized using MALDI‐TOF‐MS analysis. The sequence obtained from MALDI‐TOF‐MS showed significant similarity with the capsular polysaccharide biosynthesis protein (CapD) of Staphylococcus aureus through comparative modeling approach using ROBETTA server. Identification of responsible fragments for homodimer formation was performed using comparative modeling and substrate binding domain through C‐terminus matching of this new lipase with the CapD of Staphylococcus aureus was executed. Thus, the experimental coupled molecular modeling postulated a structure–activity relationship of lipase from S. arlettae JPBW‐1, a potential candidate for detergent, leather, pulp, and paper industries.     

Comparative analyses of three legume species reveals conserved and unique root extracellular proteins

Increasing evidence suggests that root extracellular proteins are involved in interactions between roots and their soil environment. In the present study, exudates released by 6‐day‐old roots of the three legume species white lupin (Lupinus albus), soybean (Glycine max), and cowpea (Vigna sinensis) were collected under axenic conditions, and their constitutively secreted proteomes were analyzed. Between 42 and 93 unique root extracellular proteins with 2 or more different peptide fragments per protein were identified by LC‐MS/MS. Functional annotation of these proteins classified them into 14–16 different functional categories. Among those 14 homologous proteins were identified in at least two legume species. Among the unique proteins, 58 in white lupin, 85 in soybean, and 31 in cowpea were specific for each plant species, and many of them were classified in the same functional categories. Interestingly, in contrast to soybean and cowpea, two protein bands of approximately 16 and 30 kDa were present on the SDS‐PAGE gel of white lupin. The identification of these bands revealed a class III chitinase and a thaumatin‐like protein. Both belong to the class of pathogenesis‐related proteins. The results imply that root extracellular proteins play important roles in the cross‐talk between plant roots and the rhizosphere.     

Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome

A common problem encountered when performing large‐scale MS proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). The proteome of M. pneumoniae cells, grown in defined media, was analyzed by MS. An initial search with both Mascot and a species‐specific NCBInr database with common contaminants (NCBImpn), resulted in around 79% of the acquired spectra not having an assignment. The percentage of non‐assigned spectra was reduced to 27% after re‐analysis of the data with the PEAKS software, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. Nonetheless, 33 413 spectra with assigned amino acid sequences could not be mapped to any NCBInr database protein sequence. Approximately, 1% of these unassigned peptides corresponded to PTMs and 4% to M. pneumoniae protein variants (deamidation and translation inaccuracies). The most abundant peptide sequence variants (Phe‐Tyr and Ala‐Ser) could be explained by alterations in the editing capacity of the corresponding tRNA synthases. About another 1% of the peptides not associated to any protein had repetitions of the same aromatic/hydrophobic amino acid at the N‐terminus, or had Arg/Lys at the C‐terminus. Thus, in a model system, we have maximized the number of assigned spectra to 73% (51 453 out of the 70 040 initial acquired spectra). All MS data have been deposited in the ProteomeXchange with identifier PXD002779 ( http://proteomecentral.proteomexchange.org/dataset/PXD002779 ).     

Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha2‐Macroglobulins From Serum

Secreted phosphoprotein‐24 kDa (Spp24) binds cytokines of the bone morphogenetic protein/transforming growth factor‐β (BMP/TGFβ) superfamily and is one of the most abundant serum phosphoproteins synthesized by the liver. Little is known about how Spp24 binding affects BMP signal transduction and osteoblastic differentiation or how this labile protein is transported from the liver to remote tissues, such as bone. When Spp24 was administered to W‐20‐17 mesenchymal stem cells with rhBMP‐2, short‐term Smad1/5 phosphorylation was inhibited, intermediate‐term alkaline phosphatase (ALP) induction was blunted, and long‐term mineralization was unaffected. This supports the hypothesis that Spp24 proteolysis restricts the duration of its regulatory effects, but offers no insight into how Spp24 is transported intact from the liver to bone. When Spp24 was immunopurified from serum and subjected to native PAGE and Western blotting, a high molecular weight band of >500 kDa was found. Under reducing SDS–PAGE, a 24 kDa band corresponding to monomeric Spp24 was liberated, suggesting that Spp24 is bound to a complex linked by disulfide bonds. However, such a complex cannot be disrupted by 60 mM EDTA under non‐reducing condition or in purification buffers containing 600 mM NaCl and 0.1% Tween‐20 at pH 2.7–8.5. LC–MS/MS analysis of affinity‐purified, non‐reducing SDS–PAGE separated, and trypsin digested bands showed that the Spp24 was present in a complex with three α₂‐macroglobulins (α₂‐macroglobulin [α₂M], pregnancy zone protein [PZP] and complement C3 [C3]), as well as ceruloplasmin and the protease inhibitor anti‐thrombin III (Serpin C1), which may protect Spp24 from proteolysis. J. Cell. Biochem. 114: 378–387, 2013. © 2012 Wiley Periodicals, Inc.     

Copper stress‐induced changes in leaf soluble proteome of Cu‐sensitive and tolerant Agrostis capillaris L. populations

Changes in leaf soluble proteome were explored in 3‐month‐old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1–50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO₄ spiked‐nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2‐DE (linear 4–7 pH gradient). Analysis of CCB‐stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC‐MS/MS. In both populations, Cu excess impacted both light‐dependent (OEE, cytochrome b6‐f complex, and chlorophyll a‐b binding protein), and ‐independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin‐NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S‐containing amino‐acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 ( http//proteomecentral.proteomexchange.org/dataset/PXD001930 ).     

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two‐dimensional gel electrophoresis

Introduction – A variety of sample preparation protocols for plant proteomic analysis using two‐dimensional gel electrophoresis (2‐DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. Objective – This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2‐DE. Methodology – Four sample preparation methods were tested: (1) phenol extraction and methanol–ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid–acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS–PAGE (1‐DE) and 2‐DE. Fifteen selected protein spots were trypsinised and analysed by matrix‐assisted laser desorption/ionisation time‐of‐flight tandem mass spectrometry (MALDI‐TOF‐MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Results – Methods number 3 and 4 resulted in large quantities of protein with good 1‐DE separation and were chosen for 2‐DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. Conclusion – The described sample preparation method allows the proteomic analysis of papaya leaves by 2‐DE and mass spectrometry (MALDI‐TOF‐MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

ICPLQuant – A software for non‐isobaric isotopic labeling proteomics

The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope‐coded protein label (ICPL)‐labeled peptides on the MS level during LC‐MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time‐consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS‐identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker.     

2‐D PAGE and MS analysis of proteins from formalin‐fixed,paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.     

About three‐fourths of mouse proteins unexpectedly appear at a low position of SDS‐PAGE,often as additional isoforms,questioning whether all protein isoforms have been eliminated in gene‐knockout cells or organisms

Most genes in evolutionarily complex genomes are expressed to multiple protein isoforms, but there is not yet any simple high‐throughput approach to identify these isoforms. Using an oversimplified top‐down LC–MS/MS strategy, we detected, around the 26‐kD position of SDS‐PAGE, proteins produced from 782 genes in a Cdk4?/? mouse embryonic fibroblast cell line. Interestingly, only 213 (27.24%, about one‐fourth) of these 782 genes have their proteins with a theoretical molecular mass (TMM) 10% smaller or larger than 26 kD, that is, between 23 and 29 kD, the range set as allowed variation in SDS‐PAGE. These 213 proteins are considered as the wild type (WT). The remaining three‐fourths includes proteins from 66 (9.44%) genes with a TMM smaller than 23 kD and proteins from 503 (64.32%, nearly two‐thirds) genes with a TMM larger than 29 kD; these proteins are categorized into a larger‐group or a smaller‐group, respectively, for their appearance at a higher or lower position of SDS‐PAGE. For instance, at this 26‐kD position we detected proteins from the Rps27a, Snrpf, Hist1h4a, and Rps25 genes whose proteins' TMM is 8.6, 9.7, 11.4, and 13.7 kD, respectively, and detected proteins from the Plelc1 and Prkdc genes, whose largest isoform is 533.9 and 471.1 kD, respectively. We extrapolate that many of those proteins migrating unexpectedly in SDS‐PAGE may be isoforms besides the WT protein. Moreover, we also detected a Cdk4 protein in this Cdk4?/? cell line, thus wondering whether some of other gene‐knockout cells or organisms show similar incompleteness of the knockout.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: occurrence in the air and clinical significance

Trichoderma harzianum, a filamentous fungus, is being widely used as a potential biopesticide. The potential of this fungus in causing skin sensitization, however, was poorly investigated as yet. The objective of this study was to monitor the occurrence of T. harzianum in the air and to explore its skin sensitizing potential. Seasonal periodicity of T. harzianum was studied for the years 2002–2004 by an Andersen air sampler. The skin sensitizing potential of T. harzianum extract was studied in 389 patients with suspected respiratory allergy by skin prick test (SPT) and specific IgE level was determined by ELISA. SDS–PAGE and immunoblotting were also performed. T. harzianum colony count varied from 3.69 to 134.88 CFU m⁻³ with the peak achieved in February. Relative humidity was found to be a significant (P < 0.05) factor predicting the occurrence of T. harzianum in the air. Positive skin reaction (wheal diameter ≥ 3 mm) was observed in 105 patients (26.99%). T. harzianum crude extract was resolved in 18 protein bands (12–72 kDa) on SDS–PAGE (12% gel) including two IgE-binding protein bands (21 and 32 kDa). T. harzianum can be considered an important inhalant allergen.