Low Abundant N-linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites.

Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307-309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel's initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by (18)O/PNGase F as well as by various endoglycosidases, and LC-MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ).

Study of isobaric grape seed proanthocyanidins by MALDI-TOF MS.

Positive matrix-assisted laser desorption ionization (MALDI) spectra of grape seed raw extracts show the signals of putative proanthocyanidin (PA) oligomers, including those of several sodium and potassium adducts with the same nominal molecular weight. As a result, the MALDI time-of-flight profiles are characterized by several isobaric signals from different PAs. The presence of isobaric PA adducts was studied by sodium and potassium exchange experiments on the seed extracts of six grape varieties. This approach was effective in differentiating PAs with isobaric signals, and 15 different PAs were identified in the MALDI spectra of all samples.

Chemical aspects of the primary ionization mechanisms in matrix-assisted laser desorption ionization.

It has been proposed that the primary ionization mechanism occurring in matrix-assisted laser desorption ionization (MALDI) experiments originates from the presence, in the solid-state matrix-analytes sample, of matrix dimers. These species are formed by the interaction of carboxylic groups present in the matrix molecules with the formation of strong hydrogen bonds. Theoretical calculations proved that the laser irradiation of these structures leads to one or two H-bridge cleavages, giving rise to an "open" dimer structure or to disproportionation with the formation of MH(+) and [M-H](-) species. The ions so formed can be considered highly effective in their reaction with analyte ions, leading to their protonation (or deprotonation). To achieve further evidence for these proposals, in the present study the energetics of the reactions of ions from different aromatic carboxylic acids with two amino acids (glycine and lysine) and three multipeptides (gly-gly, gly-gly-gly and gly-gly-gly-gly) was investigated. The lowest ∆G values were obtained for 2,5- dihydroxybenzoic acid, widely employed as the MALDI matrix. Also, for p-nitrobenzoic acid the reaction is slightly exothermic, while for the other aromatic carboxylic acids derivatives positives values of ∆G are present.

Protein glycation in diabetes as determined by mass spectrometry.

Diabetes is a common endocrine disorder characterized by hyperglycemia leading to nonenzymatic glycation of proteins, responsible for chronic complications. The development of mass spectrometric techniques able to give highly specific and reliable results in proteome field is of wide interest for physicians, giving them new tools to monitor the disease progression and the possible complications related to diabetes, as well as the effectiveness of therapeutic treatments. This paper reports and discusses some of the data pertaining protein glycation in diabetic subjects obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). The preliminary studies carried out by in vitro protein glycation experiments show clear differences in molecular weight of glycated and unglycated proteins. Then, the attention was focused on plasma proteins human serum albumin (HSA) and immunoglobulin G (IgG). Enzymatic degradation products of in vitro glycated HSA were studied in order to simulate the in vivo enzymatic digestion of glycated species by the immunological system leading to the highly reactive advanced glycation end-products (AGEs) peptides. Further studies led to the evaluation of glycated Apo A-I and glycated haemoglobin levels. A different MALDI approach was employed for the identification of markers of disease in urine samples of healthy, diabetic, nephropathic, and diabetic-nephropathic subjects.

On the Primary Ionization Mechanism(s) in Matrix-Assisted Laser Desorption Ionization.

A mechanism is proposed for the first step of ionization occurring in matrix-assisted laser desorption ionization, leading to protonated and deprotonated matrix (Ma) molecules ([Ma + H](+) and [Ma - H](-) ions). It is based on observation that in solid state, for carboxyl-containing MALDI matrices, the molecules form strong hydrogen bonds and their carboxylic groups can act as both donors and acceptors. This behavior leads to stable dimeric structures. The laser irradiation leads to the cleavage of these hydrogen bonds, and theoretical calculations show that both [Ma + H](+) and [Ma - H](-) ions can be formed through a two-photon absorption process. Alternatively, by the absorption of one photon only, a heterodissociation of one of the O-H bonds can lead to a stable structure containing both cationic and anionic sites. This structure could be considered an intermediate that, through the absorption of a further photon, leads to the formation of matrix ions. Some experiments have been performed to evaluate the role of thermal ionization and indicate that its effect is negligible. Some differences have been observed for different matrices in the formation of analyte molecule (M) ion [M + H](+), [M - H](-), M(+•), and [M - 2H](-•), and they have been explained in terms of ionization energies, pKa values, and thermodynamic stability.

Seed oil triglyceride profiling of thirty-two hybrid grape varieties.

Triglyceride profile of seed oil samples from 32 hybrid grape varieties not studied before was investigated. A new method for the analysis of triacylglycerols (TAGs) has been developed based on the direct infusion in the electrospray ionization (ESI) source and employing tetrahydrofuran/methanol/water (85:10:5 v|v|v) as solvent; the formation of [M + Na](+) ions in high yield has been observed. TAGs were identified by ESI-tandem mass spectrometry analysis, and the matrix-assisted-laser-desorption-ionization and time-of-flight profile of samples was determined. Six were the principal TAGs identified in seed oil: trilinolein (LLL) was the most abundant (43%), followed by dilinoleoyl-oleoylglycerol (LOL, 23%), and dilinoleoyl-palmitoylglycerol (LPL, 15%). Compounds present in lower concentration were LSL and LOO (11%), LOP (6%), and LSP (2%). Compared with seed oils produced from V. Vinifera grapes, some significant differences in the relative abundances of TAGs were found, in particular hybrid grape seed oils showed higher LOL and lower LPL content, respectively. Among the samples studied, a particularly high content of LLL (rich in unsaturated fatty acids) was found in seed oils from two red varieties.

ESI-MS and MALLS analysis of quaternary structure of molluscan hemocyanins.

The understanding of the function of macromolecular complexes is mainly related to a precise knowledge of their structure. Recently, the development of suitable mass spectrometric techniques (electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)) and multi-angle laser light scattering has enabled mass determination of native complexes and of their subunits. By these techniques, the structure and association/dissociation behavior of huge molecules of molluscan Octopus vulgaris, Sepia officinalis and Rapana venosa have been characterized. Molecular masses of the native and dissociated molecule of cephalopodan Hcs O. vulgaris (3545 and 359.3 kDa, respectively) and S. officinalis (4134 and 443.8 kDa, respectively) revealed that only one type subunit organizes their molecules, while the presence of two isoforms with different masses (422.8 and 400.0 kDa) has been determined for gastropodan R. venosa Hc, aggregated into didecamers. The difference of their structural subunits was also established after limited proteolysis with TPCK-trypsin. Eight functional units (FUs) with masses of ~ 50 kDa were isolated from both subunits of RvH and isoform of Sepia officinalis, while seven FUs were purified from OvH. Further characterization of proteins by ESI-mass spectrometry (MS) and MALDI-MS, methods gave insights into post-translational modifications such as glycosylation. Glycosylation of O. vulgaris and S. officinalis Hcs was suggested based on the differences (11.6 and 40.0 kDa, respectively) between the masses measured by ESI-MS and those calculated by their gene sequences.

Investigation on the role of pneumatic aspects in electrospray, desorption electrospray surface ionization and surface activated chemical ionization.

This review reports the results of some studies carried out by us on the role of pneumatic aspects in electrospray and desorption electrospray surface ionization, with the aim to propose some relevant aspects of the mechanisms involved in these ionization methods. Electrospray ion sources, with the exception of the nano- electrospray source, operate with the concurrent action of a strong electrical field and a supplementary coaxial gas flow. The electrical field is responsible for electrospraying of the analyte solution but the use of a coaxial gas flow leads to a significant increase of the analyte signal and allows the use of higher solution flows. However, by employing capillary voltages much lower than those necessary to activate the electrospray phenomenon, analyte ions are still observed and this indicates that different mechanisms must be operative for ion production. Under these conditions, ion generation could take place from the neutral pneumatically sprayed droplet by field-induced droplet ionization. Also in the case of desorption electrospray ionization (DESI), and without any voltage on the spraying capillary as well as on the surface of interest, ions of analytes present on the surface become detectable and this shows that desorption/ionization of analytes occurs by neutral droplets impinging the surface. Consequently, the pneumatic effect of the impinging droplets plays a relevant role, and for these reasons the method has been called pneumatic assisted desorption (PAD). Some analogies existing between PAD and surface activated chemical ionization (SACI), based on the insertion of a metallic surface inside an atmospheric pressure chemical ionization source operating without corona discharge, are discussed.

APC I1307K mutations and forkhead box gene (FOXO1A): another piece of an interesting correlation.

PURPOSE: Germline nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene are found in approximately 90% of individuals affected by familial adenomatous polyposis (FAP) and a genotype-phenotype relationship has been observed. Missense mutations have also been found in a few cases, even if their role in FAP is still unknown. An association between a missense mutation, APC I1307K, and the risk of sporadic colorectal cancer (CRC) has been reported. In order to improve the knowledge about the genetic effect of APC I1307K on the phenotype, we tried a new approach using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). EXPERIMENTAL DESIGN: An APC mutation (I1307K) was found in an index case of a non-Jewish woman and her son with attenuated familial adenomatous polyposis (A-FAP) and no family history of cancer. In order to evaluate whether the presence and abundance of the ionic species are related to the presence of cancer or the presence of mutation, comparative analyses of 11 healthy clean-colon subjects, 59 patients with CRC (stage II n=19, stage III n=23, stage IV n=17) without polyps, and 9 FAP patients, carriers of a nonsense mutation in the APC gene, were evaluated. RESULTS: Comparative analysis of serum protein profiles of the index patient and her healthy son, FAP and sporadic CRC patients, and subjects with preneoplastic lesions showed a characteristic abundance of ionic species at m/z 905, which was not present in healthy controls. Two peptides were identified from MALDI/MS/MS spectra of m/z 905 belonging to the kininogen-1 precursor and the human forkhead box protein 01A (FOXO1A). FOXO1A was present in only two subjects carrying I1307K, but not in other patients. CONCLUSIONS: Our findings seem to suggest a relationship between m/z 905, FOXO1A and the development and growth of colorectal cancer. FOXO1A fragment determination in serum with MALDI/MS might be a promising approach for early detection of colon carcinoma or for the development of targeted therapies.

The double nature of 1,5-diaminonaphthalene as matrix-assisted laser desorption/ionization matrix: some experimental evidence of the protonation and reduction mechanisms.

1,5-Diaminonaphthalene (DAN) has been described as an interesting and effective matrix for matrix-assisted laser desorption/ionization (MALDI) experiments in positive ion mode, being able to activate in-source decomposition phenomena and, when employed for the analysis of proteins containing disulphide bridge(s), being able to activate reduction processes, resulting in disulphide bridge cleavage. The mechanisms of the DAN reactivity have been studied in detail, and the results indicate that the reduction properties of the matrix are of a radical nature. In the present study the structure of the reactive species produced by DAN, responsible for its reductive properties, has been investigated by accurate mass measurements and tandem mass spectrometry (MS/MS) experiments. Contrary to what is usually observed by laser irradiation of other MALDI matrices (with the sole formation of the MH(+) ion of the matrix), DAN leads to the formation of odd-electron molecular ions M(+•) . This can be rationalized by the occurrence of two photon pooling processes, due to the low ionization energy of DAN. Thus the M(+•) ion of DAN can be considered responsible for both analyte protonation and disulphide bond reduction and some mechanisms are proposed for this behaviour.

Urinary peptides as a diagnostic tool for renal failure detected by matrix-assisted laser desorption/ionisation mass spectrometry: an evaluation of their clinical significance.

The development of new analytical methodologies related to the proteome for the evaluation of renal physiology and pathology is surely of wide interest for physicians, giving them new tools for monitoring complications associated with diabetes, such as end-stage renal disease. In the present study, the clinical significance of the urinary abundance of two peptides, SGSVIDQSRVLNLGPITR (the uromodulin precursor, m/z 1912) and IGPHypGPHypGLMGPP [present in the collagen-α-5(IV) chain precursor, m/z 1219], detected by matrix- assisted laser desorption/ionisation mass spectrometry (MALDI/MS) in microalbuminuric or nephropathic diabetic patients and in non-diabetic nephropathic patients was evaluated. A progressive increase in the abundance of the ion at m/z 1219 and a decrease in the abundance of the ion at m/z 1912 have been found in diabetic microalbuminuric, diabetic-nephropathic and nephropathic patients. Linear correlations are present between serum creatinine values and the abundances of the ions at m/z 1219 (positive correlation, r=0.3645, P<0.0001) and at m/z 1912 (negative correlation, r=-0.3053, P<0.0005). Correlations between the MALDI data and the estimated glomerular filtration rate were also found, while relationships with urinary albumin excretion were found only in sub-sets of patients. Analysis of receiver operating characteristic curves showed a sensitivity up to 96% and a specificity of up to 84% for the two ionic species, or their ratio, for distinguishing diabetic patients with different degrees of nephropathy from healthy subjects, proving that the urinary abundance of the two peptides at m/z 1219 and m/z 1912, determined with MALDI/MS, may be considered as a possible diagnostic tool for the determination of progression toward renal failure, also with the aim of monitoring kidney function, in diabetic patients.

Sieve-based device for MALDI sample preparation. III. Its power for quantitative measurements.

The solid sample inhomogeneity is a weak point of traditional MALDI deposition techniques that reflects negatively on quantitative analysis. The recently developed sieve-based device (SBD) sample deposition method, based on the electrospraying of matrix/analyte solutions through a grounded sieve, allows the homogeneous deposition of microcrystals with dimensions smaller than that of the laser spot. In each microcrystal the matrix/analyte molar ratio can be considered constant. Then, by irradiating different portions of the microcrystal distribution an identical response is obtained. This result suggests the employment of SBD in the development of quantitative procedures. For this aim, mixtures of different proteins of known molarity were analyzed, showing a good relationship between molarity and intensity ratios. This behaviour was also observed in the case of proteins with quite different ionic yields. The power of the developed method for quantitative evaluation was also tested by the measurement of the abundance of IGPP[Oxi]GPP[Oxi]GLMGPP (m/z 1219) present in the collagen-α-5(IV) chain precursor, differently expressed in urines from healthy subjects and diabetic-nephropathic patients, confirming its overexpression in the presence of nephropathy. The data obtained indicate that SBD is a particularly effective method for quantitative analysis also in biological fluids of interest.

A preliminary matrix-assisted laser desorption/ionization time-of-flight approach for the characterization of Italian lentil varieties.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used in this study to obtain protein fingerprints of seven different lentil varieties, to characterize their differences and similarities. Two different matrices have been tested in order to obtain reproducible and significant mass spectra. Extraction with water containing 0.1% of trifluoroacetic acid has been used as preparative step to obtain hydrophilic protein samples of lentil seeds. The obtained MALDI protein profiles identified clear differences between the seven studied lentil varieties. Moreover, considering the high complexity of the obtained MALDI spectra, multivariate techniques of data analysis were employed to find further classification details. These multivariate analyses confirmed the possibility of a clear classification of the seven lentil varieties, indicating that the proposed procedure can be a valid taxonomic tool, and a method to certify the origin of lentils, useful for high added value lentils (Italian lentils).

Quantification of soyasaponins I and betag in Italian lentil seeds by solid-phase extraction (SPE) and high-performance liquid chromatography-mass spectrometry (HPLC-MS).

Lentil saponins are triterpene glycosides, mainly soyasaponins I and betag (also known as VIota), with multiple health-promoting properties. This paper reports the isolation of soyasaponins I and betag from soybeans as analytical standards and the development of a new analytical procedure for quantification of their content in various cultivars of Italian lentils, by SPE-HPLC-MS. Soyasaponins I and betag were isolated from soybeans at a purity of >90% and characterized by MS/MS (ion trap) experiments. The determination of soyasaponins in lentils was performed by extraction, SPE purification, and HPLC-MS (single quadrupole) analysis; results were confirmed by MALDI-TOF experiments. Calibration curves for soyasaponin I and betag showed correlation coefficients of 0.998 and 0.997, respectively. LOD and LOQ values were 0.02 and 0.2 mg kg(-1) for soyasaponins I and 0.1 and 1 mg kg(-1) for soyasaponin betag. Recoveries calculated at a 100 mg kg(-1) fortification level ranged from 85 to 97%, with n = 10 and RSDs of <12%. In the 32 lentil samples, contents of soyasaponin I ranged from 28 to 407 mg kg(-1), whereas that of soyasaponin betag ranged from 110 to 1242 mg kg(-1).

A further investigation on a MALDI-based method for evaluation of markers of renal damage.

The validity of the urinary protein profile to characterize the pathological states of diabetic, nephropathic and diabetic-nephropathic patients was considered on the basis of previously obtained results by MALDI/MS, showing a different abundance ratio of the collagen alpha1 and alpha5 chain precursor fragments at m/z 1219 and 2049 and of the uromodulin precursor fragment at m/z 1912 observed in healthy subjects and patients; a larger number of subjects was examined and the obtained results were statistically evaluated. The p values related to the observed differences indicate that they are statistically significant when comparing all patients versus healthy controls, diabetic with normo or microalbuminuria versus nephropathic with advanced renal disease patients and diabetic with normo or microalbuminuria versus diabetic with advanced nephropathy patients. The scatter plot matrix gives evidence of the strict inverse relationship between the abundances of ions at m/z 1912 and 1219, the correlation coefficient being particularly high (r = 0.921, p < 0.001). The relationship between the true positive rate (sensitivity) and false positive rate (1-specificity) for every possible cutoff value in abundance of the considered ionic species was investigated through the receiver-operating characteristic (ROC) curve. The obtained data indicate that a good differentiation of nephropathic patients with advanced renal disease and diabetic patients with advanced nephropathy versus healthy subjects can be easily obtained by this approach.

Sieve-based device for MALDI sample preparation. II. Instrumental parameterization.

A simple device effective for matrix-assisted laser desorption/ionization (MALDI) sample preparation, based on the spraying of matrix/sample solution through a stainless steel sieve (sieve-based device, SBD), has been employed for the preparation of MALDI samples of peptides, polysaccharides and high molecular weight proteins. The instrumental parameters, mainly the sprayer voltage, solution and auxiliary gas flows, and distances between sprayer-sieve and sieve-MALDI sample holder have been optimized. The MALDI spectra obtained by laser irradiation of the microspots so obtained, exhibit resolution and sensitivity higher than those achieved by the commonly employed dried droplets (DD) method. Worth noting is the fact that, in contrast to what is observed in the DD method, identical spectra are obtained by irradiation of different areas of the MALDI sample, showing that by using the SBD the matrix/analyte ratio is constant over the entire surface.