Analysis of peptides and proteins affinity-bound to iron oxide nanoparticles by MALDI MS

Iron oxide nanoparticles modified with oleate have been employed for the extraction of peptides and proteins from aqueous solution before matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Adsorption of peptides and proteins onto the nanoparticles were mainly through electrostatic attraction and hydrophobic interaction. The analyte-adsorbed iron oxide nanoparticles could be efficiently collected from solution using a magnet. No elution step was needed. With this preconcentration strategy, the lowest detectable concentration of angiotensin I, insulin, and myoglobin in 500 microL of aqueous solution were 0.1 nM, 0.1 nM, and 10.0 nM, respectively. In addition, the nanoparticles could extract the analytes from solution with a high content of salt and surfactant, thus eliminating suppression effect during MALDI MS analysis. This method was successfully applied to concentrate the tryptic digest products of cytochrome c. In addition, the tryptic digestion of cytochrome c can be directly conducted on the iron oxide nanoparticles.     

MALDI／MS法应用于血液制品的定性质量评价

本文简述了运用基体辅助激光解吸电离质谱进行血液制品定性质量评价的方法，用此法能检出其他方法不能检出的痕量杂质蛋白。     

Carbon nanotubes as affinity probes for peptides and proteins in MALDI MS analysis

Recently, carbon nanotubes (CNTs) have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191). In a somewhat related study, we have employed CNTs produced by using NaH-treated anodic aluminum oxide (Na@AAO) as a reactive template as the assisting matrix for MALDI analysis upon the addition of high concentrations of citrate buffer. Our results indicate that the mass range can be extended to ca. 12,000 Da and that alkali metal adducts of analytes are effectively reduced. Furthermore, we have employed citric acid-treated CNTs as affinity probes to selectively concentrate traces of analytes from aqueous solutions. High concentrations of salts and surfactants in the sample solutions are also tolerated. This approach is very suitable for the MALDI analysis of small proteins, peptides, and protein enzymatic digest products.     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

An impulse-driven liquid-droplet deposition interface for combining LC with MALDI MS and MS/MS

A simple and robust impulse-driven droplet deposition system was developed for off-line liquid chromatography matrix-assisted laser desorption ionization mass spectrometry (LC-MALDI MS). The system uses a solenoid operated with a pulsed voltage power supply to generate impulses that dislodge the hanging droplets from the LC outlet directly to a MALDI plate via a momentum transfer process. There is no contact between the LC outlet and the collection surface. The system is compatible with solvents of varying polarity and viscosity, and accommodates the use of hydrophobic and hydrophilic MALDI matrices. MALDI spots are produced on-line with the separation, and do not require further processing before MS analysis. It is shown that high quality MALDI spectra from 5 fmol of pyro-Glu-fibrinopeptide deposition after LC separation could be obtained using the device, indicating that there was no sample loss in the interface. To demonstrate the analytical performance of the system as a proteome analysis tool, a range of BSA digest concentrations covering about 3 orders of magnitude, from 5 fmol to 1 pmol, were analyzed by LC-MALDI quadrupole time-of-flight MS, yielding 6 and 57% amino acid sequence coverage, respectively. In addition, a complex protein mixture of an E. coli cell extract was tryptically digested and analyzed by LC-MALDI MS, resulting in the detection of a total of 409 unique peptides from 100 fractions of 15-s intervals.     

Analysis of the binding sites with NL-101 to amino acids and peptides by HPLC/MS/MS

Collision-induced dissociation experiment demonstrated that under the same collision energy, different fragmentation pattern could help distinguish the accurate location of amino acids-NL adducts.     

Peptide peak intensities enhanced by cysteine modifiers and MALDI TOF MS

Two cysteine‐specific modifiers we reported previously, N‐ethyl maleimide (NEM) and iodoacetanilide (IAA), have been applied to the labeling of cysteine residues of peptides for the purpose of examining the enhancement of ionization efficiencies in combination with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI TOF MS). The peak intensities of the peptides as a result of modification with these modifiers were compared with the peak intensities of peptides modified with a commercially available cysteine‐specific modifier, iodoacetamide (IA). Our experiments show significant enhancement in the peak intensities of three cysteine‐containing synthetic peptides modified with IAA compared to those modified with IA. The results showed a 4.5–6‐fold increase as a result of modification with IAA compared to modification with IA. Furthermore, it was found that IAA modification also significantly enhanced the peak intensities of many peptides of a commercially available proteins, bovine serum albumin (BSA), compared to those modified with IA. This significant enhancement helped identify a greater number of peptides of these proteins, leading to a higher sequence coverage with greater confidence scores in identification of proteins with the use of IAA. Copyright © 2012 John Wiley & Sons, Ltd.     

N-terminal tagging strategy for De Novo sequencing of short peptides by ESI-MS/MS and MALDI-MS/MS

The major portion of skin secretory peptidome of the European Tree frog Hyla arborea consists of short peptides from tryptophyllin family. It is known that b-ions of these peptides undergo head-to-tail cyclization, forming a ring that can open, resulting in several linear forms. As a result, the spectrum contains multiple ion series, thus complicating de novo sequencing. This was observed in the Q-TOF spectrum of one of the tryptophyllins isolated from Hyla arborea; the sequence FLPFFP-NH₂ was established by Edman degradation and counter-synthesis. Though no rearrangements were observed in FTICR-MS and MALDI-TOF/TOF spectra, both of them were not suitable for mass-spectrometry sequencing due to the low sequence coverage. To obtain full amino acid sequence by mass spectrometry, three chemical modifications to N-terminal amino moiety were applied. They include acetylation and sulfobenzoylation of N-amino group and its transformation to 2,4,6-trimethylpyridinium by interaction with 2,4,6-trimethylpyrillium tetrafluoroborate. All three reagents block scrambling and provide spectra better than the intact peptide. Unfortunately, all of them also readily react with lysine side chain. Hence, all investigated procedures can be used to improve sequencing of short peptides, while acetylation is the recommended one. It shows excellent results, and it is plain and simple to perform. This is the procedure of choice for MS-sequencing of short peptides by manual or automatic algorithms.     

Proteome profile of zebrafish caudal fin based on one-dimensional gel electrophoresis LCMS/MS and two-dimensional gel electrophoresis MALDI MS/MS analysis

Zebrafish (Danio rerio) is the widely used vertebrate model animal for understanding the complexity of development and disease process. Zebrafish has been also extensively used in understanding the mechanism of regeneration for its extensive capability of regenerating fins and other tissues. We have analyzed the proteome profile of zebrafish caudal fin in its native state based on one-dimensional gel electrophoresis LCMS/MS and two-dimensional gel electrophoresis MS/MS analyses. A total of 417 proteins were identified as zebrafish fin tissue specific, which includes 397 proteins identified based on one-dimensional gel electrophoresis LCMS/MS analysis and 101 proteins identified based on two-dimensional gel electrophoresis MALDI MS/MS. The proteins mapped to the zebrafish fin tissue were shown to be involved in various biological activities related to development, apoptosis, signaling and metabolic process. Focal adhesion, regulation of actin cytoskeleton, cancer-related pathways, mitogen-activated protein kinase signaling, antigen processing and presentation, and proteasome are some of the important pathways associated with the identified proteome data set of the zebrafish fin.     

Fragmentation of cationized phosphotyrosine containing peptides by atmospheric pressure MALDI/Ion trap mass spectrometry

An investigation of phosphate loss from sodium-cationized phosphotyrosine containing peptide ions was conducted using liquid infrared (2.94 microm) atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) coupled to an ion trap mass spectrometer (ITMS). Previous experiments in our laboratory explored the fragmentation patterns of protonated phosphotyrosine containing peptides, which experience a loss of 98 Da under CID conditions in the ITMS. This loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. Phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence. In the absence of a basic residue, the protonated phosphotyrosine peptides do not undergo losses of HPO(3) (Delta 80 Da) nor HPO(3) + H(2)O (Delta 98 Da) in their CID spectra. However, sodium cationized phosphotyrosine containing peptides that do not contain arginine or lysine residues within their sequences do undergo losses of HPO(3) (Delta 80 Da) and HPO(3) + H(2)O (Delta 98 Da) in their CID spectra.     

Photofading of ballpoint dyes studied on paper by LDI and MALDI MS

The determination of the age of an ink entry from a questioned document is often a major problem and a controversial issue in forensic sciences. Therefore, it is important to understand the aging process of the different components found in ink. The aim of this work is to characterize the degradation processes of methyl violet and ethyl violet, two typical ballpoint dyes by using laser desorption/ionization (LDI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), and to evaluate the possible application of the method to forensic examination of documents. The mass spectrometric methods were first tested and were found to be adequate for the purpose of this work. Moreover, it is possible to analyze the dye from a stroke directly from the paper (LDI-MS), so the sample preparation is minimized. The degradation of the dyes methyl violet and ethyl violet in strokes from a ballpoint pen was studied under laboratory conditions influenced by different factors such as light, wavelength of light, heat, and humidity. Then, strokes from the same ballpoint were aged naturally in the dark or under the influence of light over one year and then analyzed. The results show that the degradation of these dyes strongly depends on light fluence. Humidity also increases degradation, which can be explained by the basicity of the paper. The influence of heat on the degradation process was found to be rather weak. It was also observed that the dyes from the ink strokes did not show significant degradation after one year of storage in the dark. In conclusion, the storage conditions of a questioned document and the initial composition of the dyes in the ink have to be known for correct interpretation of the age of an ink entry. Measurements over longer periods of time are necessary to follow the degradation of dyes exempt from light exposure. LDI was found adequate and very useful for the analysis of ballpoint dyes directly from paper without further pretreatment.     

Origin of product ions in the MS/MS spectra of peptides in a quadrupole ion trap

Stored waveform inverse Fourier transform and double resonance techniques have been used in conjunction with a quadrupole ion trap to study the dissociation patterns of peptide ions. These experiments provide insight into the origin of individual product ions in an MS/MS spectrum. Results show for a series of leucine enkephalin analogues with five amino acid residues that the b ₄ ion is the main product ion through which many other product ions arise. It was also observed that the percentage of the a ₄ product ions that are formed directly from the protonated molecule (M+H)⁺ depends on the nature of the fourth amino acid residue. In addition, it was determined that in the peptides studied here lower series b ions (e.g., b ₃ arise from direct dissociation of higher series b ions (e.g., b ₄ only about 50% of the time.     

Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich c kinase protein

A series of phosphorylated test peptides was studied by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). The extensive ECD-induced fragmentation made identification of phosphorylation sites for these peptides straightforward. The site(s) of initial phosphorylation of a synthetic peptide with a sequence identical to that of the phosphorylation site domain (PSD) of the myristoylated alanine-rich C kinase (MARCKS) protein was then determined. Despite success in analyzing fragmentation of the smaller test peptides, a unique site on the PSD for the first step of phosphorylation could not be identified because the phosphorylation reaction produced a heterogeneous mixture of products. Some molecules were phosphorylated on the serine closest to the N-terminus, and others on one of the two serines closest to the C-terminus of the peptide. Although no definitive evidence for phosphorylation on either of the remaining two serines in the PSD was found, modification there could not be ruled out by the ECD fragmentation data.     

Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.     

Label-free quantification of peptides in solution by disposable patterned hydrophilic chip based MALDI imaging

Quantification of a mixture of peptides in solution was achieved by disposable patterned hydrophilic chip based matrix-assisted laser desorption/ionization mass spectrometric imaging(MALDI MSI).Compared with other quantitative methods for peptides in solution, this method is label-free and does not require separation of the multiple components of the solution before analysis. Uniform hydrophilic spots and high mass accuracy measurements provided confident identification and quantitative analysis of imaged compounds. The linear correlation between concentration and grayscale of image in the range of 5 fmol/μ L to 1 pmol/μ L was obtained for all four peptides. Good sensitivity and excellent reproducibility were also achieved. The method expands the application of MALDI MSI from tissues to solutions.     

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have increased its application in recent years. The most common method of "peptide mass fingerprint" analysis often does not provide robust identification. Additional sequence information, obtained by post-source decay or collision induced dissociation, provides additional constraints for database searches. However, de novo sequencing by mass spectrometry is not yet common practice, most likely because of the difficulties associated with the interpretation of high and low energy CID spectra. Success with this type of sequencing requires full sequence coverage and demands better quality spectra than those typically used for data base searching. In this report we show that full-length de novo sequencing is possible using MALDI TOF/TOF analysis. The interpretation of MS/MS data is facilitated by N-terminal sulfonation after protection of lysine side chains (Keough et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136). Reliable de novo sequence analysis has been obtained using sub-picomol quantities of peptides and peptide sequences of up to 16 amino acid residues in length have been determined. The simple, predictable fragmentation pattern allows routine de novo interpretation, either manually or using software. Characterization of the complete primary structure of a peptide is often hindered because of differences in fragmentation efficiencies and in specific fragmentation patterns for different peptides. These differences are controlled by various structural parameters including the nature of the residues present. The influence of the presence of internal Pro, acidic and basic residues on the TOF/TOF fragmentation pattern will be discussed, both for underivatized and guanidinated/sulfonated peptides.     

Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b- and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 160/18O water during digestion has been explored and de novo sequences for a number of peptides have been obtained.     

Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV

Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement membranes. Human collagen type IV was modified via incubation with 200 mM NaNO(2) (pH=7.38) for one week at 37(o)C. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C(12) RP column. Site specificity was determined from tandem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical R(t), lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(IV)Y1081. These sites are located in the triple-helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV.     

Detection of Delta9-tetrahydrocannabinolic acid A in human urine and blood serum by LC-MS/MS

Delta9-tetrahydrocannabinolic acid A (Delta9-THCA-A) is the precursor of Delta9-tetrahydrocannabinol (Delta9-THC) in hemp plants. During smoking, the non-psychoactive Delta9-THCA-A is converted to Delta9-THC, the main psychoactive component of marihuana and hashish. Although the decarboxylation of Delta9-THCA-A to Delta9-THC was assumed to be complete--which means that no Delta9-THCA-A should be detectable in urine and blood serum of cannabis consumers--we found Delta9-THCA-A in the urine and blood serum samples collected from police controls of drivers suspected for driving under the influence of drugs (DUID). For LC-MS/MS analysis, urine and blood serum samples were prepared by solid-phase extraction. Analysis was performed with a phenylhexyl column using gradient elution with acetonitrile. For detection of Delta9-THCA-A, the mass spectrometer (MS) (SCIEX API 365 triple-quadrupole MS with TurboIonSpray source) was operated in the multiple reaction monitoring (MRM) mode using the following transitions: m/z357 --> 313, m/z357 --> 245 and m/z357 --> 191. Delta9-THCA-A could be detected in the urine and blood serum samples of several cannabis consumers in concentrations of up to 10.8 ng/ml in urine and 14.8 ng/ml in serum. The concentration of Delta9-THCA-A was below the Delta9-THC concentration in most serum samples, resulting in molar ratios of Delta9-THCA-A/Delta9-THC of approximately 5.0-18.6%. Only in one case, where a short elapsed time between the last intake and blood sampling is assumed, the molar ratio was 18.6% in the serum. This indicates differences in elimination kinetics, which need to be investigated in detail.