Biocytin hydrazide--a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin-biotin technology

Biocytin hydrazide (BCHZ), a new, water-soluble, long-chained, biotin-containing hydrazide, was synthesized and used for the selective nonradioactive detection of glycoconjugates. Procedures were developed for labeling glycoconjugates on blots. The method involves either chemical (periodate-induced) or enzymatic (via galactose oxidase) oxidation of glycoconjugates, the resultant aldehyde groups are then labeled with biocytin hydrazide, followed by interaction with an avidin-based enzyme probe. Since the biotin-containing reagent is a relatively small, charged molecule, the primary labeling step may be carried out on intact cells and on membrane preparations as well as on blotted samples. On blots, the labeling pattern was similar for both periodate- and galactose oxidase-induced biotinylation procedures. In contrast, periodate-induced labeling of either erythrocyte membranes or cells (prior to blotting) produced an altered labeling pattern. Combined enzyme-induced biotinylation of membranes or cells resulted in a pattern similar to that observed for the direct staining of blots. Using galactose oxidase on human erythrocyte membranes, the procedure was sensitive enough to selectively label the Band 3 lactosaminoglycoprotein.     

3-(N-Maleimido-propionyl)biocytin: a versatile thiol-specific biotinylating reagent

A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) biocytin (MPB), was synthesized and used to biotinylate various proteins via native or artificially induced sulfhydryl groups. In combination with appropriate avidin- or streptavidin-conjugated markers (i.e., fluorescent, enzyme-conjugated, electron-dense, etc.), MPB essentially constitutes a universal, multipurpose, thiol-specific probe. The reagent could be used to detect protein SH groups on dot blots with sensitivities in the femtomole range. The labeling was very specific for sulfhydryl groups or reduced S-S bonds; proteins lacking free SH groups were unlabeled by this method. Due to the long spacer between the biotinyl group and the reactive maleimide, improved adsorption of biotinylated proteins to avidin columns was achieved. An SH-containing enzyme (beta-galactosidase) was biotinylated with MPB, and the resultant biotinylated enzyme could be used as an efficient histochemical probe. The use of this reagent is recommended to biotinylate proteins which contain nonessential SH groups or which can be easily thiolylated prior to reaction with MPB.     

Selective labeling of sulfhydryls and disulfides on blot transfers using avidin-biotin technology: studies on purified proteins and erythrocyte membranes

Various strategies for the use of 3-(N-maleimido-propionyl) biocytin (MPB) as a general label for distinguishing between protein sulfhydryls and disulfides on blot transfers are presented. In the first approach, endogenous SH groups in proteins were labeled directly with MPB. For disulfide staining, endogenous sulfhydryls were blocked with N-ethylmaleimide, disulfides were then reduced with mercaptoethanol, and the newly formed SH groups were labeled with MPB. In this approach, all treatments were performed in vitro, and, between steps, excess reagent was removed by dialysis. The MPB-labeled proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (in the presence of mercaptoethanol), the labeled proteins were transferred to nitrocellulose, and the blotted proteins were detected by avidin-biotin technology. In the second approach, MPB treatment was performed directly on blot transfers. For SH labeling, proteins were subjected to SDS-PAGE in the absence of mercaptoethanol, thus retaining the status of endogenous sulfhydryl and disulfide groups. For S-S labeling, proteins were treated with N-ethylmaleimide in vitro and then subjected to SDS-PAGE in the presence of mercaptoethanol, such that endogenous sulfhydryls were blocked and endogenous disulfides were converted to SH groups. Subsequent treatments and washings were performed on blots. In the third approach, immobilized proteins (i.e., in artificial systems or in natural systems such as membrane preparations or intact cells) were treated essentially as described in the first approach, except that washings were carried out by centrifugation. In vitro treatments were performed before SDS-PAGE (carried out in the presence of mercaptoethanol) and subsequent blot transfer. The relative merits of the three strategies are discussed.     

p-Diazobenzoyl biocytin--a new biotinylating reagent for the labeling of tyrosines and histidines in proteins

A new biotin-containing reagent, p-diazobenzoyl biocytin (DBB), has been developed for labeling tyrosines and histidines in proteins. The reagent has used to label these residues in both model proteins and erythrocyte membrane proteins on dot blots and blot transfers. In some cases, sub-nanogram levels of individual proteins could be detected. The utility of DBB as a versatile alternative to biotin-containing N-hydroxysuccinimide esters for the general labeling of proteins is discussed.     

Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione.

Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of activities of the cells. Qualitative and quantitative analyses of sulfhydryl groups in the sperm membrane were performed by fluorescence microscopy, fluorimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidofluoresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Triton-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) biocytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydryl groups after cooling at 4 degrees C and freezing/thawing. Cryopreservation of spermatozoa changed the distribution pattern of sulfhydryl groups on sperm surface measured with fluorescence microscopy using 5-iodoacetamidofluoresceine. The percentage of spermatozoa labeled at the level of the mid-piece decreased by 50 and 90% after cooling and freezing/thawing, respectively. Spin labeling studies showed a 15% faster rotational diffusion (mobility) of sulfhydryl containing proteins in the membrane of frozen/thawed spermatozoa as compared to that of fresh spermatozoa. Addition of glutathione, reduced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented the effects of freezing/thawing, such as higher exposure of sulfhydryl groups, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to the cryoprotectant reduced by 35% the loss of motility of spermatozoa undergoing a freezing/thawing cycle. We concluded that cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservation medium.     

Improved biocytin labeling and neuronal 3D reconstruction

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.     

A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies

Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state or time-resolved fluorescence resonance energy transfer (FRET) detection of the kinetics of protein folding require relatively large quantities (approximately 10-100 mg) of site-specific doubly labeled protein samples. Engineered cysteine residues are common targets for labeling of proteins. The challenge here is to develop methods for selective modification of one of two reactive sulfhydryl groups in a protein molecule. A general systematic procedure for selective labeling of each of two cysteine residues in a protein molecule was developed, using Escherichia coli adenylate kinase (AKe) as a model protein. Potential sites for insertion of cysteine residues were selected by examination of the crystal structure of the protein. A series of single-cysteine mutants was prepared, and the rates of the reaction of each engineered cysteine residue with a reference reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] were determined. Two-cysteine mutants were prepared by selection of pairs of sites for which the ratio of this reaction rate constant was high (>80). The conditions for the selective labeling reaction were optimized. In a first cycle of labeling, the more reactive cysteine residue was labeled with a fluorescent probe (donor). The second probe was attached to the less reactive site under unfolding conditions in the second cycle of labeling. The doubly and singly labeled mutants retained full enzymatic activity and the capacity for a reversible folding-unfolding transition. High yields (70-90%) of the preparation of the pure, site-specific doubly labeled AK mutant were obtained. The procedure described herein is a general outline of procedures, which can meet the double challenge of both site specificity and large-scale preparation of doubly labeled proteins.     

Newly synthesized proteins in seminiferous intertubular and intratubular compartments of the rat testis

Two-dimensional gel electrophoresis combined with autoradiography and Western blot procedures have been used to characterize newly synthesized proteins in testicular intertubular fluid (TIF) and seminiferous tubular fluid (SNF). Fluids were collected following in vivo and in vitro intratesticular injection of [35S]methionine into control and hypophysectomized adult rats. A discrete number of [35S]methionine-labeled proteins were detected within TIF and SNF. Their presence and relative abundance varied according to in vivo and in vitro labeling conditions. While two major blood plasma proteins, albumin and transferrin, were radioactively labeled after in vivo labeling, these two proteins were insignificantly labeled in samples collected after in vitro labeling. Three acidic proteins, possibly secreted by Sertoli cells (Mr = 72,000, 45,000 and 35,000), were more abundant in TIF samples collected after in vitro [35S]methionine labeling than after in vivo labeling. Incubated seminiferous tubules and TIF of hypophysectomized rats showed a decrease in [35S]methionine-labeling intensity of the Mr = 72,000 acidic protein, possibly reflecting changes in the seminiferous epithelium caused by pituitary hormonal deprivation. Autoradiographs of TIF and most remarkably, of SNF, showed many protein spots that suggested cell breakage and leakage during sample collection. Results of this study suggest that most albumin and transferrin found in TIF and SNF have an extratesticular origin and that proteins secreted by the Sertoli cell can gain access to both TIF and SNF.     

Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters

Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytometry 10:11-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.     

A propolis extract and the labeling of blood constituents with technetium-99m

Since ancient times propolis has been employed for many human purposes because to their favourable properties. Blood constituents labeled with technetium-99m (99mTc) have been used in nuclear medicine procedures. Some authors have reported that synthetic or natural drugs can interfere with the labeling of blood constituents with 99mTc. The aim of this work was to evaluate the action of a propolis extract on the labeling of blood elements with 99mTc. Samples of whole blood of male Wistar rats were incubated in sequence with an aqueous propolis extract at different concentrations, stannous chloride and 99mTc, as sodium pertechnetate. Blood samples were centrifuged to separate plasma and blood cells, soluble and insoluble fractions of plasma and blood cells were also separated after precipitation in trichloroacetic acid solution and centrifugation. The radioactivity was counted and the percentage of incorporated radioactivity (%ATI) for each fraction was calculated. The data obtained showed that the aqueous propolis extract used decreased significantly the %ATI in plasma proteins at higher concentration studied. Results suggest that at high concentration the constituents of this extract could alter the labeling of plasma proteins competing with same binding sites of the 99mTc on the plasma proteins or acting as antioxidant compounds.     

Fluorescent labeling of cysteinyl residues to facilitate electrophoretic isolation of proteins suitable for amino-terminal sequence analysis

A protein labeling procedure which enables detection of subpicomole quantities of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gels is described. Proteins are rendered fluorescent by reduction of disulfide bonds with dithiothreitol followed by alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) or 5-iodoacetamido-fluorescein. Labeling is performed prior to electrophoresis, thus eliminating the need for staining with dyes and destaining after electrophoresis. As little as 375 fmol (25 ng) of prelabeled bovine serum albumin can be readily visualized after electrophoresis. Bands are still visible after electrophoretic transfer to nitrocellulose. Simultaneous labeling of proteins in complex mixtures is possible using this technique. This includes cysteine containing proteins of disrupted Newcastle disease virus. The magnitudes of the molecular weight increases which occur upon labeling reflect the cysteine contents of proteins. The mode of chemical modification for the prelabeling procedure was chosen because of its compatibility with analytical techniques, such as amino acid analysis, peptide mapping, or sequence analysis, which may be applied to the protein after electroelution from SDS-acrylamide gels. It replaces the need for reduction and carboxymethylation prior to these analytical procedures. Protein-sequence analysis of prelabeled bovine serum albumin, including samples electroeluted from SDS-acrylamide gels, has justified the choice of this method to facilitate isolation of proteins for sequence analysis. Equivalent sequence data were obtained with reduced bovine serum albumin S-alkylated with iodoacetic acid or 5-I-AEDANS.     

A simple fluorescence labeling method for studies of protein oxidation, protein modification, and proteolysis

Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve (3)H or (14)C methylation, which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid precipitation. Alternative labeling methods have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, (3)H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well suited to studying increased proteolytic susceptibility after protein modification, because the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite and is stable over time and to extremes of pH, temperature (even boiling), freeze-thaw, mercaptoethanol, and methanol.     

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-H]Thymidine in a Hypertrophic Lake

The incorporation of [methyl-H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-H]thymidine studies of aquatic bacterial production.     

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.     

Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches

The role of thioredoxin in wheat starchy endosperm was investigated utilizing two proteomic approaches. Thioredoxin targets were isolated from total KCl-soluble extracts of endosperm and flour and separated by 2-DE following (1) reduction of the extract by the NADP/thioredoxin system and labeling the newly generated sulfhydryl (SH) groups with monobromobimane (mBBr), and, in parallel, (2) trapping covalently interacting proteins on an affinity column prepared with mutant thioredoxin h in which one of the active site cysteines was replaced by serine. The two procedures were complementary: of the total targets, one-third were observed with both procedures and one-third were unique to each. Altogether 68 potential targets were identified; almost all containing conserved cysteines. In addition to confirming known interacting proteins, we identified 40 potential thioredoxin targets not previously described in seeds. A comparison of the results obtained with young endosperm (isolated 10 days after flowering) to those with mature endosperm (isolated 36 days after flowering) revealed a unique set of proteins functional in processes characteristic of each developmental stage. Flour contained 36 thioredoxin targets, most of which have been found in the isolated developing endosperm.     

Global Protein Differential Expression Profiling of Cerebrospinal Fluid Samples Pooled from Chinese Sporadic CJD and non-CJD Patients

The shotgun proteomic based on the approach of tandem mass tag (TMT) labeling has received increasing attention for neuroproteomics analysis and becomes an effective tool for the identification and quantification of a large number of proteins for the purpose of revealing key proteins involved in the neuronal dysfunction and an inflammatory response associated with neurodegenerative disorders. To assess the potential expression difference of proteins in cerebrospinal fluids (CSF) between Creutzfeldt–Jakob disease (CJD) and non-CJD patients, the pooled CSF samples from 39 Chinese probable sporadic CJD (sCJD) patients and from 52 non-CJD cases were comparably analyzed with the methodology of TMT labeling and RP-RP-UPLC-MS/MS. Totally, 437 possible proteins were identified in the tested CSF specimen, among them, 49 proteins with 95 % confidence interval. Differential assays showed among those 49 CSF proteins, 12 were upregulated and 13 were downregulated significantly in the sCJD compared to non-CJD. The most affected pathway of the differential expression proteins in CSF of sCJD was complement and coagulation cascade. Western blots for six selected changed proteins in the pooled CSF samples revealed the similar altering profiles in the groups of sCJD and non-CJD as proteomics. Furthermore, CSF samples from 24 CJD patients and 24 non-CJD patients were randomly selected and subjected individually into the Western blots of an increased protein (phosphoglycerate mutase 1) and a decreased one (alpha-1-antichymotrysin), which also confirmed the altering tendency of these identified proteins. Those data indicate that proteomic assay of CSF is a powerful technique not only for selection of the potential biomarkers for the development of diagnostic tool of CJD but also for supplement of useful scientific clues for understanding the CSF homeostasis during the pathogenesis of prion diseases.     

Determination of biocytin

Biocytin (epsilon-N-[d-biotinyl]-L-lysine) is generally undetected in serum and other body fluids of normal healthy individuals in view of the ubiquitous distribution of biotinidase. It has been suggested that biocytin may be present in serum and urine of patients with inherited biotinidase deficiency. We have developed a noncompetitive assay for biocytin based on its interaction with avidin. Biocytin can be determined in biological samples containing both biotin and biocytin. Biotin from such samples is removed by an anion-exchange resin and the biocytin is determined by the avidin-binding assay. The effect of above-normal levels of biotin in the sample on the assay of biocytin is discussed.     

Development of bifunctional photoactivatable benzophenone probes and their application to glycoside substrates

Photoaffinity labeling is used to covalently attach ligands to macromolecules to determine their spatial arrangement and structure. Benzophenone (BP) groups are widely used for covalent photoaffinity labeling and for probing protein interactions. We developed bifunctional BP photoactivatable derivatives using three different general chemical approaches. In addition to the photoaffinity reactivity of the BP, these derivatives contain an additional group: A radioactive tracer for biological studies, or an N-ethylmaleimide group as an additional crosslinker, or a biotin group to be used during purification and characterization of probe-protein complexes using the high-affinity biotin-avidin interaction. A model series of photoaffinity labeling probes was synthesized based on the arbutin ligand. These compounds can be used as probes to study the arbutin binding site of microbial beta-glucoside transporters by photolabeling residues in its vicinity. The second functionality provides additional options for studying proteins and binding sites. The probes were developed using different methodologies: (i) a diazotation reaction; (ii) protecting group methodology; and (iii) solid-phase synthesis. These procedures are general and provide a simple and versatile approach for synthesizing bifunctional BP ligands, as demonstrated here on arbutin.     

Labeling of antibodies by in situ modification of thiol groups generated from selenol-catalyzed reduction of native disulfide bonds

A new method for labeling antibodies which involves selenol-catalyzed reduction of native disulfide bonds in antibodies to generate thiol groups, which then are labeled using thiol-reactive reagents, is described. The reduction and labeling steps of this rapid procedure are carried out in one vessel, without requiring any separation step to remove the reductant before labeling. It results in a quantitative and homogenous incorporation of about seven labeled groups per antibody molecule in less than 5 min. All reagents used are commercially available-selenocystamine (catalyst precursor), dithiothreitol or tris(2-carboxyethyl)phosphine (reductant), and thiol-reactive labeling reagents such as biotin-poly(ethylene oxide)-maleimide. This method is broadly applicable for labeling proteins such as immunoglobulins with reducible disulfide bonds, whose reduction and labeling does not result in a significant loss of activity. Biotinylated murine antibodies (anti-phosphotyrosine and anti-EGF receptor) prepared by this reduced-disulfide labeling method perform comparably or better than amino-group biotinylated antibodies in applications such as enzyme-linked immunosorbent assay, immunohistochemistry, and immunoprecipitation. This reduced-disulfide labeling method is superior to amino-group labeling methods because it is not inhibited by the presence of amines in solution, as demonstrated by the biotinylation of an antibody in a hybridoma culture supernatant containing amino acids and serum proteins.     

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.