COMPLX: a computer algorithm for the detection of protein-ligand and other macromolecular complexes in mass spectra

A new algorithm has been designed and tested to identify protein, or any other macromolecular, complexes that have been widely reported in mass spectral data. The program takes advantage of the appearance of multiply charged ions that are common to both electrospray ionization and, to a lesser extent, matrix-assisted laser desorption/ionization (MALDI) mass spectra. The algorithm, known as COMPLX for the COMposition of Protein-Ligand compleXes, is capable of identifying complexes for any protein or macromolecule with a binding partner of molecular mass up to 100 000 Da. It does so by identifying ion pairs present in a mass spectrum that, when they share a common charge, have an m/z value difference that is an integer fraction of a ligand or binding partner molecular mass. Several additional criteria must be met in order for the result to be ranked in the output file including that all m/z values for ions of the protein or complex have progressively lower values as their assigned charge increases, the difference between the m/z values for adjacent charge states (z, z + 1) decrease as the assigned charge state increases, and the ratio of any two m/z values assigned to a protein or complex is equal to the inverse ratio of their charge. The entries that satisfy these criteria are then ranked according to the appearance of ions in the mass spectrum associated with the binding partner, the length of a continuous series of charges across any set of ions for a protein and complex and the lowest error recorded for the molecular mass of the ligand or binding partner. A diverse range of hypothetical and experimental mass spectral data were used to implement and test the program, including those recorded for antibody-peptide, protein-peptide and protein-heme complexes. Spectra of increasing complexity, in terms of the number of ions input, were also successfully analysed in which the number of input m/z values far exceeds the few associated with a macromolecular complex. Thus the program will be of value in a future goal of proteomics, where mass spectrometry already plays a central role, for the direct analysis of protein and other associations within biological extracts.     

Automated deconvolution and deisotoping of electrospray mass spectra

Electrospray ionization (ESI) of peptides and proteins produces a series of multiply charged ions with a mass/charge (m/z) ratio between 500 and 2000. The resulting mass spectra are crowded by these multiple charge values for each molecular mass and an isotopic cluster for each nominal m/z value. Here, we report a new algorithm simultaneously to deconvolute and deisotope ESI mass spectra from complex peptide samples based on their mass-dependent isotopic mean pattern. All signals corresponding to one peptide in the sample were reduced to one singly charged monoisotopic peak, thereby significantly reducing the number of signals, increasing the signal intensity and improving the signal-to-noise ratio. The mass list produced could be used directly for database searching. The developed algorithm also simplified interpretation of fragment ion spectra of multiply charged parent ions.     

A computer algorithm for the identification of protein interactions from the spectra of masses (PRISM)

A new algorithm is reported to assist with the identification of protein interaction domains by comparing pairs of MALDI mass spectra recorded for protein digests treated with a binding partner versus an untreated control. Known as PRISM, for protein interactions from the spectra of masses, the algorithm imports m/z versus peak area data directly from a pair of MALDI mass spectra recorded for the control and reaction sample. The algorithm is shown to be able to successfully identify antigenic determinants for protein antigens within mixed protein digests. The algorithm has general utility for the comparative analysis of differences within any two mass spectra of any type and is easily implemented using a simple, intuitive graphical user interface (GUI).     

Drug binding revealed by tandem mass spectrometry of a protein-micelle complex

The protein-micelle complex formed between the protein EmrE and the lipid dodecylmaltoside has been examined by mass spectrometry. The results show that despite the unfavorable hydrophobic environment in the mass spectrometer it is possible to preserve protein submicelle complexes in the gas phase. The peaks assigned to the submicelle complexes are broad in nature and consistent with a heterogeneous distribution of lipid molecules attached to the protein complex. As such, the spectrum cannot be interpreted. To simplify this complexity we used a tandem mass spectrometry procedure in which discrete m/z values are isolated from the peak and subjected to collision-induced dissociation. These spectra reveal clusters of DDM molecules as well as sequential release of TPP+ and EmrE from the complex as the collision cell voltage is raised. Taken together, the results provide direct evidence for drug binding within a relevant gas-phase protein-micelle complex.     

若干环戊二烯基(N,N-二苄基氨荒酸)锆衍生物的质谱研究

本文通过电子轰击谱,并借助于同位素丰度分析和亚稳跃迁数据,阐述了标题衍生物(MeCp)Zr(S_2CN(C_7H_7)_2)_3(Ⅰ)、CpZr(S_2CN(C_7H_7)_2)_3(Ⅱ)、(MeCp)_2ZrCl(S_2CN(C_7H_7)_2)(Ⅲ)和Cp_2ZrCl(S_2CN(C_7H_7)_2)(Ⅳ)的质谱断裂途径。对于配合物(Ⅰ)和(Ⅱ),可看到从分子离子中首先丢失N,N-二苄基氨荒酸配体或该配体的部分碎片离子(C_7H_7~+),构成两个配合物的主要断裂途径。而丢失Cp或MeCp配体,在两种配合物质谱中则有差别。一般地说,Cp较MeCp易于丢失。这可能由于MeCp比Cp有较强给电子能力,因而使配体与金属间键得以加强。对于配合物(Ⅲ)和(Ⅳ),上述关系不明显。在配合物(Ⅳ)中,通过同位素丰度分析,指明m/z 462和m/z 463两种不同离子组成的两簇峰相互重叠,构成观察到的一簇叠加同位素峰。     

Tandem mass spectrometric investigation of acylpolyamines of spider venoms and their <Superscript>15</Superscript>N-labeled derivatives

The fragmentation mechanism of the acylpentamine toxins 1-4 found in the venom of the spider Agelenopsis aperta has been investigated in detail. To identify the origin of the two doublets of unexpected fragment ions at m/z 129/112 and m/z 115/98, three synthetic 15N-labeled analogs 5-7 have been prepared and subjected to CID fragmentation on a triple quadrupole mass spectrometer. It appears that the unexpected doublet of fragment ions arises from an internal portion of the polyamine backbone after either a transaminative Zip reaction or a sequential fragmentation of the quasi-molecular ion. The second option has been proven by in-source CID experiments. The detailed knowledge of acylpentamine fragmentation mechanisms is essential for the correct characterization of isomeric compounds, particularly for coeluting compounds within complex mixtures such as spider venoms.     

Accurate mass precursor ion data and tandem mass spectrometry identify a class I human leukocyte antigen A*0201-presented peptide originating from vaccinia virus

We have used accurate mass precursor ion data generated on a hybrid linear-ion trap-Fourier transform ion cyclotron resonance mass spectrometer to augment tandem mass spectrometry (MS/MS) data generated on two different instrument types. Results from these experiments have allowed us for the first time to identify a naturally processed peptide presented by a class I human leukocyte antigen allele (HLA-A*0201) that was isolated from B cells infected by live vaccinia, the viral agent of the smallpox vaccine. The accurate mass data, in conjunction with MS/MS data, was able to identify the sequence IVIEAIHTV (aa 187-195) from the protein thymidylate kinase of vaccinia, distinguishing it from a similar sequence IVLEAIAEH: a "self-peptide" from the human protein phospholipase Cbeta3. Accurate mass data for the doubly charged species from the naturally processed and presented peptide was 497.8006, which was within 0.8 ppm of the calculated m/z of 497.8002, while being -37.3 ppm from the calculated m/z (497.7820) of the second-ranked peptide sequence IVLEAIAEH. Accurate mass data ranged from less than 0.1 to 1.2 ppm for other peptides identified in this sample. A BLAST search shows this sequence, IVIEAIHTV, is conserved in the same protein of a number of other orthopoxviruses, including the variola (smallpox) virus. Additionally, accurate mass data were able to uncover a false positive search result that was not distinguished by scoring of the match to the MS/MS data.     

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.     

Using ion purity scores for enhancing quantitative accuracy and precision in complex proteomics samples

To accurately determine the quantitative change of peptides and proteins in complex proteomics samples requires knowledge of how well each ion has been measured. The precision of each ions' calculated area is predicated on how uniquely it occupies its own space in m/z and elution time. Given an initial assumption that prior to the addition of the "heavy" label, all other ion detections are unique, which is arguably untrue, an initial attempt at quantifying the pervasiveness of ion interference events in a representative binary SILAC experiment was made by comparing the centered m/z and retention time of the ion detections from the "light" variant to its "heavy" companion. Ion interference rates were determined for LC-MS data acquired at mass resolving powers of 20 and 40 K with and without ion mobility separation activated. An ion interference event was recorded, if present in the companion dataset was an ion within ± its Δ mass at half-height, ±15 s of its apex retention time and if utilized by ±1 drift bin. Data are presented illustrating a definitive decrease in the frequency of ion interference events with each additional increase in selectivity of the analytical workflow. Regardless of whether the quantitative experiment is a composite of labeled samples or label free, how well each ion is measured can be determined given knowledge of the instruments mass resolving power across the entire m/z scale and the ion detection algorithm reporting both the centered m/z and Δ mass at half-height for each detected ion. Given these measurements, an effective resolution can be calculated and compared with the expected instrument performance value providing a purity score for the calculated ions' area based on mass resolution. Similarly, chromatographic and drift purity scores can be calculated. In these instances, the error associated to an ions' calculated peak area is estimated by examining the variation in each measured width to that of their respective experimental median. Detail will be disclosed as to how a final ion purity score was established, providing a first measure of how accurately each ions' area was determined as well as how precise the calculated quantitative change between labeled or unlabelled pairs were determined. Presented is how common ion interference events are in quantitative proteomics LC-MS experiments and how ion purity filters can be utilized to overcome and address them, providing ultimately more accurate and precise quantification results across a wider dynamic range.     

Nitrite oxidation in ion chromatography-electrospray ionization-tandem mass spectrometry (IC-ESI-MS/MS)

Nitrite anions are formed in the human body and in the natural environment as intermediate chemical compounds during the reduction of nitrate, a ubiquitous anthropogenic contaminant introduced into the environment primarily through fertilizer use. Multiple reaction monitoring (MRM) in ion chromatography-electrospray ionization-tandem mass spectrometry (IC-ESI-MS/MS) is a promising new technique for quantifying and confirming the identity of anions in complex aqueous mixtures. In this article, we present the results of a short investigation devised to: (1) compare the signal generated by the MRM transitions for nitrite with those for nitrate, (2) isolate the source of the signal from these MRM transitions occurring within the IC-ESI-MS/MS instrument and (3) assess the relationship between the observed MRM signals for nitrite. The MRM transitions used in this study were m/z 62 (NO(3)(-))→m/z 46 (NO(2)(-)) and m/z 46 (NO(2)(-))→m/z 46 (NO(2)(-)). Results of the investigation revealed the association of both MRM transitions with the nitrite chromatographic peak, indicating the occurrence of nitrite oxidation to nitrate at the ESI interface before the first quadrupole. Calibrations for both MRM signals, as well as their sum, were found to be linear. However, the ratio of m/z 62→m/z 46 to m/z 46→m/z 46 (indicating an extent of oxidation) ranged from 35 to 56% over a nitrite concentration range of 10 to 100 ppm, showing no clear trend associated with concentration.     

ToF-SIMS Analysis of Adsorbed Proteins: Principal Component Analysis of the Primary Ion Species Effect on the Protein Fragmentation Patterns

In time-of-flight secondary ion mass spectrometry (ToF-SIMS), the choice of primary ion used for analysis can influence the resulting mass spectrum. This is because different primary ion types can produce different fragmentation pathways. In this study, analysis of single-component protein monolayers were performed using monatomic, tri-atomic, and polyatomic primary ion sources. Eight primary ions (Cs(+), Au(+), Au(3) (+), Bi(+), Bi(3) (+), Bi(3) (++), C(60) (+)) were used to examine to the low mass (m/z < 200) fragmentation patterns from five different proteins (bovine serum albumin, bovine serum fibrinogen, bovine immunoglobulin G and chicken egg white lysozyme) adsorbed onto mica surfaces. Principal component analysis (PCA) processing of the ToF-SIMS data showed that variation in peak intensity caused by the primary ions was greater than differences in protein composition. The spectra generated by Cs(+), Au(+) and Bi(+) primary ions were similar, but the spectra generated by monatomic, tri-atomic and polyatomic primary ion ions varied significantly. C(60) primary ions increased fragmentation of the adsorbed proteins in the m/z < 200 region, resulting in more intense low m/z peaks. Thus, comparison of data obtained by one primary ion species with that obtained by another primary ion species should be done with caution. However, for the spectra generated using a given primary ion beam, discrimination between the spectra of different proteins followed similar trends. Therefore, a PCA model of proteins created with a given ion source should only be applied to datasets obtained using the same ion source. The type of information obtained from PCA depended on the peak set used. When only amino acid peaks were used, PCA was able to identify the relationship between proteins by their amino acid composition. When all peaks from m/z 12-200 were used, PCA separated proteins based on a ratio of C(4)H(8)N(+) to K(+) peak intensities. This ratio correlated with the thickness of the protein films and Bi(1) (+) primary ions produced the most surface sensitive spectra.     

Realistic modeling of ion cloud motion in a Fourier transform ion cyclotron resonance cell by use of a particle-in-cell approach

Using a 'Particle-In-Cell' approach taken from plasma physics we have developed a new three-dimensional (3D) parallel computer code that today yields the highest possible accuracy of ion trajectory calculations in electromagnetic fields. This approach incorporates coulombic ion-ion and ion-image charge interactions into the calculation. The accuracy is achieved through the implementation of an improved algorithm (the so-called Boris algorithm) that mathematically eliminates cyclotron motion in a magnetic field from digital equations for ion motion dynamics. It facilitates the calculation of the cyclotron motion without numerical errors. At every time-step in the simulation the electric potential inside the cell is calculated by direct solution of Poisson's equation. Calculations are performed on a computational grid with up to 128 x 128 x 128 nodes using a fast Fourier transform algorithm. The ion populations in these simulations ranged from 1000 up to 1,000,000 ions. A maximum of 3,000,000 time-steps were employed in the ion trajectory calculations. This corresponds to an experimental detection time-scale of seconds. In addition to the ion trajectories integral time-domain signals and mass spectra were calculated. The phenomena observed include phase locking of particular m/z ions (high-resolution regime) inside larger ion clouds. A focus was placed on behavior of a cloud of ions of a single m/z value to understand the nature of Fourier transform ion cyclotron resonance (FTICR) resolution and mass accuracy in selected ion mode detection. The behavior of two and three ion clouds of different but close m/z was investigated as well. Peak coalescence effects were observed in both cases. Very complicated ion cloud dynamics in the case of three ion clouds was demonstrated. It was found that magnetic field does not influence phase locking for a cloud of ions of a single m/z. The ion cloud evolution time-scale is inversely proportional to magnetic field. The number of ions needed for peak coalescence depends quadratically on the magnetic field.     

A new derivatization strategy for the analysis of phosphopeptides by precursor ion scanning in positive ion mode

Although numerous strategies have been devised to analyze protein phosphorylation, an abundant intracellular protein modification, there is still a need for different methods for the analysis of this modification. A method to both detect and localize the phosphorylation within a protein/peptide is especially required. In this paper, a new strategy is described, which makes use of beta-elimination/Michael addition reactions to introduce a functional group at the original site of phosphorylation, which gives rise to a dimethylamine-containing sulfenic acid derivative with a unique m/z value. This enables the detection of the phosphorylated species within peptide mixtures by sensitive and specific precursor ion scanning in positive ion mode. Working under acidic conditions in positive ion mode has the added advantage that subsequent normal peptide sequencing for the exact localization can be performed. No other peptide derived fragment ion is observed at the m/z value of the sulfenic acid derivative formed, thus specific precursor ion experiments can also be carried out on instruments with low fragment ion resolution and lends itself to LC-MS/MS approaches when skimmer fragmentation routines or triple quadrupole mass spectrometers are used.     

Electrophilic aromatic substitution and single-electron transfer (SET) by the phenylium ion in the gas phase: characterization of a long-lived SET intermediate

Gas-phase mass spectrometric studies and calculations were performed for the reaction of naked phenylium ion with several benzene halides. From these reactions, the molecular ion for biphenyl as the predominant product was obtained only from the reaction of phenylium ions with iodobenzene and bromobenzene. Furthermore, through the collision-induced dissociation (CID) of the ion at m/z 281, the only dissociation observed is the loss of a phenyl radical, which indicates that a single-electron transfer (SET) mechanism might have occurred within the reaction. Additionally, according to the comparison between the CID experiments of those isomeric compounds of the sigma-complexes and the CID experiment of the ion at m/z 281 captured in the ion trap, we have also defined the captured ion at m/z 281 as an SET-intimate ion pair rather than those of sigma-complexes or the diphenyliodonium.     

Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.     

Electrospray ionization tandem mass spectrometric studies to probe the interaction of Cu(II) with amoxicillin简

This study describes the application of electrospray ionization mass spectrometry(ESI-MS) to investigate copper ion interaction with amoxicillin. ESI mass spectra of Cu–amoxicillin complexes show complex ions at m/z 828, 792, 753, 731, 428, 388 and 366 corresponding to [63Cu+(2A-H)+2H2 O]+, [63Cu+(2A-H)]+, [2A+Na]+, [2A+H]+, [63Cu+(A-H)]+, [A+Na]+and [A+H]+(where A = amoxicillin). Based on the observed m/z values of Cu–amoxicillin complex ions, it is found that the Cu–amoxicillin ratios are 1:1 and 1:2, and the copper ions exhibited three feasible coordination numbers(2, 4 and 6) with amoxicillin complexes. The structures and coordination numbers of copper–amoxicillin complex ions were probed from their collisionally activated dissociation(CAD) spectra. Based on these results, it is confirmed that the copper ions could form stable tetrahedral and octahedral complexes with amoxicillin. This study validates the applicability of ESI-MS for probing copper–amoxicillin complex ions.     

Automated charge state determination of complex isotope-resolved mass spectra by peak-target Fourier transform

This study describes a new algorithm for charge state determination of complex isotope-resolved mass spectra. This algorithm is based on peak-target Fourier transform (PTFT) of isotope packets. It is modified from the widely used Fourier transform method because Fourier transform may give ambiguous charge state assignment for low signal-to-noise ratio (S/N) or overlapping isotopic clusters. The PTFT algorithm applies a novel "folding" strategy to enhance peaks that are symmetrically spaced about the targeted peak before applying the FT. The "folding" strategy multiplies each point to the high-m/z side of the targeted peak by its counterpart on the low-m/z side. A Fourier transform of this "folded" spectrum is thus simplified, emphasizing the charge state of the "chosen" ion, whereas ions of other charge states contribute less to the transformed data. An intensity-dependent technique is also proposed for charge state determination from frequency signals. The performance of PTFT is demonstrated using experimental electrospray ionization Fourier transform ion cyclotron resonance mass spectra. The results show that PTFT is robust for charge state determination of low S/N and overlapping isotopic clusters, and also useful for manual verification of potential hidden isotopic clusters that may be missed by the current analysis algorithms, i.e., AID-MS or THRASH.     

MS3 using the collision cell of a tandem mass spectrometer system

We report the feasibility of multistage fragmentation in combination with a fast background subtraction method, yielding the equivalent of MS3. The first quadrupole selects an ion of interest, and the ion is axially accelerated into Q2 to generate fragment ions. Subsequent stages of mass selection and fragmentation are obtained by quadrupolar resonant excitation within the Q2 collision cell. The fragments are analyzed downstream by either a resolving quadrupole or a time-of-flight (TOF) mass spectrometer, and multistage spectra are obtained by subtraction (MS(n) - MS(n-1)) for n = 3 or 4. We discuss the characterization of this method, including product ion arrival times, fragmentation efficiencies, and ion selectivity. We report accurate TOF mass spectra of background-subtracted MS3 for protonated molecules reserpine (m/z 609), bosentan (m/z 1552), and taxol (m/z 854).     

Automated reduction and interpretation of

Here a fully automated computer algorithm is applied to complex mass spectra of peptides and proteins. This method uses a subtractive peak finding routine to locate possible isotopic clusters in the spectrum, subjecting these to a combination of the previous Fourier transform/Patterson method for primary charge determination and the method for least-squares fitting to a theoretically derived isotopic abundance distribution for m/z determination of the most abundant isotopic peak, and the statistical reliability of this determination. If a predicted protein sequence is available, each such m/z value is checked for assignment as a sequence fragment. A new signal-to-noise calculation procedure has been devised for accurate determination of baseline and noise width for spectra with high peak density. In 2 h, the program identified 824 isotopic clusters representing 581 mass values in the spectrum of a GluC digest of a 191 kDa protein; this is >50% more than the number of mass values found by the extremely tedious operator-applied methodology used previously. The program should be generally applicable to classes of large molecules, including DNA and polymers. Thorough high resolution analysis of spectra by Horn (THRASH) is proposed as the program's verb.     

The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument

The effect of elevating the pressure in the interface region of an electrospray ionization orthogonal time-of-flight mass spectrometer on the ion intensity of different noncovalent protein assemblies has been investigated. Elevating the pressure in the interface region generally led to an enhanced detection of high m/z ions. The optimum pressure was found to be dependent on the m/z value of the ions. This pressure effect should be carefully addressed when relating ion abundance in the mass spectra to solution phase abundance of noncovalent protein assemblies.