Two-dimensional Electrophoretic Separation Research Articles

Mucopolysaccharidoses diagnosis in the era of enzyme replacement therapy in Egypt

BackgroundUndegraded glycosaminoglycans (GAGs) induced by deficiency of enzymes are the primary cause of mucopolyscchardoses. Mucopolysacchardoses (MPS) are a group of rare lysosomal storage diseases (LSD). The quantification of a specific enzymatic activity is needed for accurate diagnosis. The objectives of this work were: first, to continue the study of mucopolysacchardoses disease in Egypt after the start of using the enzyme replacement therapy (ERT). Second, to define the commonest types among our population after 18 years experience with the disease. Third, to compare the different MPS types’ distribution, diagnosed after the start of the ERT, to identify the impact of using ERT on the number and type of diagnosed patients. MethodUrinary GAGs were measured for all referred cases followed by two-dimensional electrophoretic separation for cases with high levels of GAGs; the specific enzyme activity was assayed for each type depending on the abnormal electrophoretic pattern obtained. Clinically suspected cases of Morquio syndrome were directly subjected to measuring the specific enzyme. ResultsOut of 1448 suspected cases, 622 (42.9%) MPS patients were diagnosed revealing the following distribution: MPS I (172, 27.7%), MPS II (57, 9.1%), MPS III [(177, 28.5%: 134 type B and 43 types A, C or D)], MPS IVA (124, 19.9%), MPS VI (90, 14.5%) and MPS VII (2, 0.3%). MPS III was the most commonly diagnosed type followed by MPS I and MPS IVA. MPS IVA represented the most common type receiving treatment, followed by MPS I, MPS II and MPS VI. ConclusionThe presence of treatment encouraged the affected families and physicians to seek diagnosis. MPS III was the commonest type among our studied group after 7 years of diagnosis, while MPS IVA was the commonest type receiving treatment.

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization. Graphical abstract Two-dimensional capillary electrophoresis system hyphenated with mass spectrometry for the characterization of CE (SDS)-separated proteins. As first dimension, a generic and high MS-interfering CE (SDS) separation is performed for size separation. After heart-cut transfer of the unknown CE (SDS) protein peak, via a four-port nanoliter valve to a volatile electrolyte system as second dimension, interference-free mass spectrometric data of separated mAb fragments and soybean proteins are obtained.

2-D Western blotting for evaluation of antibodies developed for detection of host cell protein.

Recombinant proteins generated for therapeutic use must be substantially free of residual host cell protein (HCP). The presence of host cell protein (HCP) is usually assayed by ELISA using a polyclonal antibody mixture raised against a population of proteins derived from the host cell background. This antibody should recognize as high a proportion as possible of the potential HCPs in a given sample. A recommended method for evaluating the assay involves two-dimensional electrophoretic separation followed by Western blotting.We present here a method using commercial anti-HCP antibody and samples derived from Chinese Hamster Ovary (CHO) cells. The 2-D electrophoresis procedure gives highly reproducible spot patterns and entire procedure can be completed in less than 2 days. Software analysis enables the straightforward generation of percent coverage values for the antibody when used to probe HCP-containing samples.

Cu2+-assisted two dimensional charge-mass double focusing gel electrophoresis and mass spectrometric analysis of histone variants

Abundant isoforms and dynamic posttranslational modifications cause the separation and identification of histone variants to be experimentally challenging. To meet this need, we employ two-dimensional electrophoretic gel separation followed by mass spectrometric detection which takes advantage of the chelation of Cu2+ with amino acid residues exposed on the surfaces of the histone proteins. Acid-extracted rat liver histones were first mixed with CuSO4 solution and then separated in one dimension with triton–acid–urea (TAU) gel electrophoresis and in a second dimension using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separations result from both the changes in charge and mass upon Cu2+ chelation. Identities of each separated gel bands were obtained by using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). It was found that the migration of H3 histone isoforms of rat liver is markedly affected by the use of Cu2+ ions.

Fifteen years experience: Egyptian metabolic lab

BackgroundInborn errors of metabolism (IEM) are single gene disorders responsible for abnormalities in the synthesis or catabolism of proteins, carbohydrates and fats by means of defective enzymes or transport proteins which results in a block of the metabolic pathway and accumulation of metabolites in different tissues. This study shows the most common diagnosed inherited inborn errors of metabolism among the Egyptian population. Prior to 1995, the diagnosis of inherited metabolic disorders in Egypt was very limited and diagnosed mainly on clinical suspicion. In 1995, The Biochemical Genetics Unit at The National Research Centre has been established as a part of The Human Genetics Department and later on in 2003 it was developed into The Biochemical Genetics Department by applying advanced techniques and equipments and providing early diagnosis for the metabolic disorders which led to better outcome in our patients. Material and methodsWe have retrospectively reviewed a total of 12,148 cases suspected to have inborn errors of metabolism (IEM) with different age groups. They had been referred from several diagnostic centers and hospitals in Egypt to The Department of Biochemical Genetics at The National Research Centre. The diagnosis of these disorders was confirmed by qualitative determination of amino acid profile, quantitative determination of phenylalanine and galactose levels using dried blood spots (DBSs), quantitative determination of urinary glycosaminoglycans (GAGs), two-dimensional electrophoretic separation of GAGs in urine and the assay for lysosomal enzymes activities in plasma and leukocytes. ResultsOut of the total number of cases; 1041 (8.6%) patients were proved to have metabolic disorders. Those patients were classified as: 722 patients (69.4%) with lysosomal storage disorders, 302 patients (29%) with amino acid disorders and 17 patients (1.6%) with galactosemia. ConclusionThis study illustrates the experience of the reference metabolic lab in Egypt over 15years. The lab began metabolic disorder screening by using simple diagnostic techniques like thin layer chromatography and colored tests in urine which by time updated and upgraded the methods to diagnose a wide range of disorders. This study shows the most common diagnosed inherited inborn errors of metabolism among the Egyptian population.

Proteomic characterization of the venom and transcriptomic analysis of the venomous gland from the Mexican centipede Scolopendra viridis.

Animal venoms of Scolopendra species have been scarcely studied, although they have been reported to contain several bioactive compounds, some of which with potential therapeutic interest. The Mexican centipede S. viridis contains a powerful venom, capable of inflicting immediate effects on their preys. This communication is focused on the identification and description of a proteomic and transcriptomic analysis of the protein components of this venom. Several amino acid sequences similar to reported enzymes are the principal components in the S. viridis venom, but also a low number of toxins were identified. This knowledge should contribute to the understanding of the pharmacological effects caused by bites of this centipede species.

Biochemical diagnosis of mucopolysaccharidoses over 11 years

Aim of the study The aim of the study was to perform biochemical laboratory diagnosis of mucopolysaccharidoses (MPS) among clinically suspected patients who referred to our department and to find out the frequency of each type of MPS among the studied patients. Patients and methods The study included 1249 patients who referred to our Biochemical Genetics Laboratory during the last 11 years for the diagnosis or exclusion of MPS. Each patient was subjected to quantitative determination of total urinary glycosaminoglycans (GAGs). Patients with high concentrations and patients clinically suspected to have MPS type-IV (104 patients of the 1249) were subjected to electrophoretic separation of GAGs in urine. The activity of the specific enzymes was fluorometrically assayed in the patients proved to have MPS by electrophoresis and in those suspected clinically to have Morquio syndrome (MPS type-IV) even with normal levels of total urinary GAGs or normal electrophoretic separation. Results Of the 1249 patients screened for MPS, 548 (43.9%) patients had elevated total GAGs in urine. Using two-dimensional electrophoretic separation of GAGs extracted from urine in patients with high total GAGs and in patients suspected clinically to have Morquio syndrome, 278 (22.3% of the total 1249) patients proved to be affected by MPS. The specific enzyme assay in the 278 positive patients revealed the following distribution: MPS type-I (n=79) (28.5% of the 278), MPS type-II (n=46) (16.5% of the 278), MPS type-III B (n=18) (6.5% of the 278), MPS type-III, probable A, C, or D undetermined (n=25) (9% of the 278), MPS type-IV A (n=39) (13.9% of the 278), and MPS type-VI (n=71) (25.5% of the 278). Conclusion Quantitative determination of total GAGs in urine is a simple procedure to select patients for electrophoretic separation of GAGs. However, enzymatic assay is mandatory to confirm the MPS type, especially with the presence of enzyme replacement therapy for types-I, II, and VI and for prenatal diagnosis in coming pregnancies. The commonest MPS disorders in this study were MPS type-I followed by type-VI and then type-II.

Column coupling isotachophoresis–capillary electrophoresis with mass spectrometric detection: Characterization and optimization of microfluidic interfaces

Two-dimensional electrophoretic separations are one of the most promising tools for the continuously growing needs of different bioanalytical fields such as proteomics and metabolomics. In this work we present the design and the implementation of a two-dimensional electrophoretic separation coupled to mass spectrometry. We started our work studying the sample transfer characteristics of different microfluidic interfaces compatible with capillary coupling for two-dimensional electrophoretic separations. These junctions are aimed at method decoupling and sample transfer in a modular two-dimensional electrophoretic separation system. In order to perform the characterization of the interfaces, we carried out capillary electrophoresis experiments and numerical simulations using three cationic compounds under different flow conditions. The comparison of the experimental and simulation results enables us to clearly define the desirable characteristics of interfaces in order to achieve method orthogonality with lossless sample transfer in a two-dimensional separation system. Finally, we present a glass microfluidic chip as interface for the implementation of a novel hybrid modular system for performing two-dimensional electrophoretic separations involving isotachophoresis and capillary electrophoresis. In this setup we include mass spectrometric and contactless capacitively coupled conductivity detection to monitor the separation process. We demonstrate the ability of the setup to be used as a flexible analysis tool by performing preconcentration, separation, detection and identification of four different human angiotensin peptides.

Pretty But Imperfect

**Objective:** To search for biologic markers in the Guillain--Barré syndrome (GBS), we studied CSF samples from patients with GBS and neuropathy of various etiologies for the presence of 14-3-3 protein. **Methods:** CSF samples from patients with GBS, chronic neuropathies, motor neuron disease (MND), definite sporadic Creutzfeldt--Jakob disease (sCJD), and normal control subjects were analyzed by standard immunoblot assay, using a polyclonal anti-14-3-3 antibody. CSF samples were also tested with antibodies recognizing specific isoforms of 14-3-3 proteins, either after one-dimensional or two-dimensional electrophoretic separation. **Results:** A positive 14-3-3 assay was observed in 29 of 38 patients with GBS and in 4 patients with MND and other neuropathies, including 2 subjects with vasculitic neuropathy (VN). In GBS, 14-3-3 protein was detected as early as 12 to 48 hours after disease onset and showed an isoform pattern different from that encountered in patients with noninflammatory neuropathies, VN, MND, and sCJD. Immunohistochemical studies performed in archival fatal GBS cases disclosed marked 14-3-3 expression by mononuclear inflammatory infiltrates and Schwann cells. **Conclusion:** CSF 14-3-3 assay may represent a useful biologic marker in patients with Guillain--Barré syndrome.

Re-examination of the dimerization state of leucine-rich repeat kinase 2: predominance of the monomeric form

Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene have been identified in PARK8, a major form of autosomal-dominantly inherited familial Parkinson's disease, although the biochemical properties of LRRK2 are not fully understood. It has been proposed that LRRK2 predominantly exists as a homodimer on the basis of the observation that LRRK2, with a theoretical molecular mass of 280 kDa, migrates at 600 kDa (p600 LRRK2) on native polyacrylamide gels. In the present study, we biochemically re-examined the nature of p600 LRRK2 and found that p600 LRRK2 was fractionated with a single peak at ~272 kDa by ultracentrifugation on a glycerol gradient. In addition, p600 LRRK2 behaved similarly to monomeric proteins upon two-dimensional electrophoretic separation. These results suggested a monomeric composition of p600 LRRK2 within cells. The p600 LRRK2 exhibited kinase activity as well as GTP-binding activity, and forced dimerization of LRRK2 neither upregulated its kinase activity nor altered its subcellular localization. Collectively, we conclude that the monomer form of LRRK2 is predominant within cells, and that dimerization is dispensable for its enzymatic activity.

Identification of Eps15 as Antigen Recognized by the Monoclonal Antibodies aa2 and ab52 of the Wuerzburg Hybridoma Library against Drosophila Brain

The Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies.

Protein Profile of Bacillus subtilis Spore

Natural wild-type strains of Bacillus subtilis spore is regarded as a non-pathogenic for both human and animal, and has been classified as a novel food which is currently being used as probiotics added in the consumption. To identify B. subtilis spore proteins, we have accomplished a preliminary proteomic analysis of B. subtilis spore, with a combination of two-dimensional electrophoretic separations and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF-MS). In this article, we presented a reference map of 158 B. subtilis spore proteins with an isoelectric point (pI) between 4 and 7. Followed by mass spectrometry (MS) analysis, we identified 71 B. subtilis spore proteins with high level of confidence. Database searches, combined with hydropathy analysis and GO analysis revealed that most of the B. subtilis spore proteins were hydrophilic proteins related to catalytic function. These results should accelerate efforts to understand the resistance of spore to harsh conditions.

Evaluation of Alkyne‐Modified Isoprenoids as Chemical Reporters of Protein Prenylation

Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide- or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkyne-containing analogs for chemical proteomic applications.

“Unknown Genome” Proteomics: A New NAD(P)-dependent Epimerase/Dehydratase Revealed by N-terminal Sequencing, Inverted PCR, and High Resolution Mass Spectrometry

We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23-47 and 91-118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.

Experimental and Statistical Considerations to Avoid False Conclusions in Proteomics Studies Using Differential In-gel Electrophoresis

In quantitative proteomics, the false discovery rate (FDR) can be defined as the number of false positives within statistically significant changes in expression. False positives accumulate during the simultaneous testing of expression changes across hundreds or thousands of protein or peptide species when univariate tests such as the Student's t test are used. Currently most researchers rely solely on the estimation of p values and a significance threshold, but this approach may result in false positives because it does not account for the multiple testing effect. For each species, a measure of significance in terms of the FDR can be calculated, producing individual q values. The q value maintains power by allowing the investigator to achieve an acceptable level of true or false positives within the calls of significance. The q value approach relies on the use of the correct statistical test for the experimental design. In this situation, a uniform p value frequency distribution when there are no differences in expression between two samples should be obtained. Here we report a bias in p value distribution in the case of a three-dye DIGE experiment where no changes in expression are occurring. The bias was shown to arise from correlation in the data from the use of a common internal standard. With a two-dye schema, where each sample has its own internal standard, such bias was removed, enabling the application of the q value to two different proteomics studies. In the case of the first study, we demonstrate that 80% of calls of significance by the more traditional method are false positives. In the second, we show that calculating the q value gives the user control over the FDR. These studies demonstrate the power and ease of use of the q value in correcting for multiple testing. This work also highlights the need for robust experimental design that includes the appropriate application of statistical procedures.

Simultaneous detection of N‐terminal fragment ions in a protein mixture using a ruthenium(II) complex

Use of a bis(terpyridine)ruthenium(II) derivative as an N-terminal labeling reagent resulted in the simultaneous detection and individual determination of all the N-terminal fragments of the proteins in a mixture without requiring any separation. All of the N-termini of the guanidinated proteins were labeled selectively by the ruthenium complex (<Ru>-CO-labeling). After chymotryptic digestion, the fragments were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and post-source decay (PSD). The <Ru>-CO moiety exclusively enhanced N-terminal fragment ions in mass spectra and enabled easy N-terminal sequencing. In a mixture containing three different proteins (lysozyme, ubiquitin, and insulin), all of the N-terminal fragment ions labeled with the ruthenium complex were found to produce uniformly intense peaks without the detection of the other unlabeled fragments. The N-terminal sequences of these ions were determined individually by PSD analysis. Application to unknown proteins from Thermus thermophilus HB8 with two-dimensional electrophoretic separation resulted in the successful determination of the N-terminal sequence and easy identification of the target protein.

Mite-related bacterial antigens stimulate inflammatory cells in rosacea

Patients with papulopustular rosacea have a higher density of Demodex folliculorum mites on their faces than normal subjects but the role, if any, of their mites in initiating inflammation is disputed. Selective antibiotics are effective in reducing the inflammatory changes of papulopustular rosacea, but their mode of action is unknown. To investigate whether a D. folliculorum-related bacterium was capable of expressing antigens that could stimulate an inflammatory immune response in patients with rosacea. A bacterium (Bacillus oleronius) was isolated from a D. folliculorum mite extracted from the face of a patient with papulopustular rosacea, and was investigated further. This bacterium produced antigens capable of stimulating peripheral blood mononuclear cells proliferation in 16 of 22 (73%) patients with rosacea but only five of 17 (29%) control subjects (P = 0.0105). This antigenic preparation was fractionated into 70 subfractions and the proteins in each fraction were visualized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Western blot analysis revealed the presence of two antigenic proteins of size 62 and 83 kDa in fractions when probing with sera from patients with rosacea. No immunoreactivity to these proteins was recorded when probing with sera from control patients. Two-dimensional electrophoretic separation was used to isolate these proteins and matrix-assisted laser desorption/ionization time-of-flight analysis was employed to identify the relevant peptides. The 62-kDa immunoreactive protein shared amino acid sequence homology with an enzyme involved in carbohydrate metabolism and signal transduction while the 83-kDa protein was similar to bacterial heat shock proteins. Antigenic proteins related to a bacterium (B. oleronius), isolated from a D. folliculorum mite, have the potential to stimulate an inflammatory response in patients with papulopustular rosacea.

Use of the arginine‐specific butanedione/phenylboronic acid tag for analysis of peptides and protein digests using matrix‐assisted laser desorption/ionization mass spectrometry

We have applied an arginine-specific labeling technique to the study of peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The reaction converts the guanidine group of the arginine side chain by reacting it with 2,3-butanedione and an arylboronic acid. Despite the general chemical lability of the tag under acidic conditions, it was possible to employ acidic matrices like alpha-cyano-4-hydroxycinnamic acid without adverse effects, using the thin-layer technique for preparation. After optimizing the method using arginine-containing model peptides--for which sensitivity down to the low fmol range was demonstrated--the procedure was applied to enzymatic digests of several model proteins in solution and to protein spots in gels obtained by two-dimensional electrophoretic separation of cell lysate samples. Information on the presence of arginine in peptides can be easily obtained from the mass spectra by the characteristic mass shift and the isotope pattern resulting from the incorporation of boron. This information might serve as a valuable additional search constraint for achieving a higher degree of confidence for protein identification by peptide mass fingerprinting.

Detection of CSF 14-3-3 protein in Guillain–Barré syndrome

To search for biologic markers in the Guillain-Barré syndrome (GBS), we studied CSF samples from patients with GBS and neuropathy of various etiologies for the presence of 14-3-3 protein. CSF samples from patients with GBS, chronic neuropathies, motor neuron disease (MND), definite sporadic Creutzfeldt-Jakob disease (sCJD), and normal control subjects were analyzed by standard immunoblot assay, using a polyclonal anti-14-3-3 antibody. CSF samples were also tested with antibodies recognizing specific isoforms of 14-3-3 proteins, either after one-dimensional or two-dimensional electrophoretic separation. A positive 14-3-3 assay was observed in 29 of 38 patients with GBS and in 4 patients with MND and other neuropathies, including 2 subjects with vasculitic neuropathy (VN). In GBS, 14-3-3 protein was detected as early as 12 to 48 hours after disease onset and showed an isoform pattern different from that encountered in patients with noninflammatory neuropathies, VN, MND, and sCJD. Immunohistochemical studies performed in archival fatal GBS cases disclosed marked 14-3-3 expression by mononuclear inflammatory infiltrates and Schwann cells. CSF 14-3-3 assay may represent a useful biologic marker in patients with Guillain-Barré syndrome.

Proteome analysis of bronchoalveolar lavage in lung diseases

The proteomic approach is complementary to genomics and enables protein composition to be investigated under various clinical conditions. Its application to the study of bronchoalveolar lavage (BAL) is extremely promising. BAL proteomic studies were initially based on two-dimensional electrophoretic separation of complex protein samples and subsequent identification of proteins by different methods. With the techniques available today it is possible to attain many different research objectives. BAL proteomics can contribute to the identification of proteins in alveolar spaces with possible insights into pathogenesis and clinical application for diagnosis, prognosis and therapy. Many proteins with different functions have already been identified in BAL. Some could be biomarkers that need to be individually confirmed by correlation with clinical parameters and validation by other methods on larger cohorts of patients. The standardization of BAL sample preparation and processing for proteomic studies is an important goal that would promote and facilitate clinical applications. Here, we review the principal literature on BAL proteomic analysis applied to the study of lung diseases.