质谱分析福尔马林固定组织的多肽谱

福尔马林固定组织时,甲醛与蛋白质多肽链的氨基酸侧链上的功能基团,特别是氨基、亚氨基、酰氨基、羟基和巯基等相结合,使蛋白分子间形成大分子网络,蛋白不再发生迁移达到固定目的[1].     

Minimization of Fragmentation and Aggregation by Laser Desorption Laser Ionization Mass Spectrometry

Measuring average quantities in complex mixtures can be challenging for mass spectrometry, as it requires ionization and detection with nearly equivalent cross-section for all components, minimal matrix effect, and suppressed signal from fragments and aggregates. Fragments and aggregates are particularly troublesome for complex mixtures, where they can be incorrectly assigned as parent ions. Here we study fragmentation and aggregation in six aromatic model compounds as well as petroleum asphaltenes (a naturally occurring complex mixture) using two laser-based ionization techniques: surface assisted laser desorption ionization (SALDI), in which a single laser desorbs and ionizes solid analytes; and laser ionization laser desorption mass spectrometry (L²MS), in which desorption and ionization are separated spatially and temporally with independent lasers. Model compounds studied include molecules commonly used as matrices in single laser ionization techniques such as matrix assisted laser desorption ionization (MALDI). We find significant fragmentation and aggregation in SALDI, such that individual fragment and aggregate peaks are typically more intense than the parent peak. These fragment and aggregate peaks are expected in MALDI experiments employing these compounds as matrices. On the other hand, we observe no aggregation and only minimal fragmentation in L²MS. These results highlight some advantages of L²MS for analysis of complex mixtures such as asphaltenes.

肽质量指纹谱鉴定蛋白质时生物信息学分析条件的优化

为了优化肽质量指纹谱(peptide mass fingerprint,PMF)鉴定蛋白质的生物信息学分析条件。将牛碳酸酐酶2(carbonic anhydrase-2,CAH2)和人热休克蛋白70s(Hsp70s)进行2-DE分离、酶解,肽段经过MALDI-TOFMS分析得到PMF数据。选择Swissprot、MSDB、NCBInr、Random等数据库和MASCOT与MS-Fit搜索引擎,以牛CAH2为模型优化搜索参数,结果表明:Swissprot是适合做蛋白PMF分析的数据库;主要参数最佳设置为:漏切位点数为1个,肽质量容错数为±1Da,同时肽质量类型选择平均分子质量比单同位素质量更便于候选蛋白的筛选。最后用人Hsp70s蛋白的PMF数据检验优化条件,结果表明,所选择的数据库及参数是可靠的。     

Relative Stability of Peptide Sequence Ions Generated by Tandem Mass Spectrometry

We report the use of unimolecular dissociation by infrared radiation for gaseous multiphoton energy transfer to determine relative activation energy (E_a,laser) for dissociation of peptide sequence ions. The sequence ions of interest are mass-isolated; the entire ion cloud is then irradiated with a continuous wave CO₂ laser, and the first order rate constant, k_d, is determined for each of a series of laser powers. Provided these conditions are met, a plot of the natural logarithm of k_d versus the natural logarithm of laser power yields a straight line, whose slope provides a measure of E_a,laser. This method reproduces the E_a values from blackbody radiative dissociation (BIRD) for the comparatively large, singly and doubly protonated bradykinin ions (nominally y ₉ and y ₉ ²⁺ ). The comparatively small sequence ion systems produce E_a,laser values that are systematic underestimates of theoretical barriers calculated with density functional theory (DFT). However, the relative E_a,laser values are in qualitative agreement with the mobile proton model and available theory. Additionally, novel protonated cyclic-dipeptide (diketopiperazine) fragmentation reactions are analyzed with DFT. FT-ICR MS provides access to sequence ions generated by electron capture dissociation, infrared multiphoton dissociation, and collisional activation methods (i.e., b _n , y _m , c _n , z _m ^• ions).     

多肽抗体免疫富集-质谱法检测肝癌患者血清多肽标志物

血清多肽是癌症诊断信息的重要来源.近年来针对其复杂多样、丰度低、易降解的特点,多种多肽组技术得到快速发展.建立并优化了检测多肽标志物的免疫质谱方法,用于检测血清中的肿瘤多肽标志物.方法的线性范围为1-40 pmol;检出限为0.5 pmol;对5 pmol合成肽 5标准品平行检测3次的相对标准偏差(RSD)为9.5%....     

Phase Correction for Absorption Mode Two-Dimensional Mass Spectrometry

Two-dimensional mass spectrometry (2D MS) is a tandem mass spectrometry method that relies on manipulating ion motions to correlate precursor and fragment ion signals. 2D mass spectra are obtained by performing a Fourier transform in both the precursor ion mass-to-charge ratio (m/z) dimension and the fragment ion m/z dimension. The phase of the ion signals evolves linearly in the precursor m/z dimension and quadratically in the fragment m/z dimension. This study demonstrates that phase-corrected absorption mode 2D mass spectrometry improves signal-to-noise ratios by a factor of 2 and resolving power by a factor of 2 in each dimension compared to magnitude mode. Furthermore, phase correction leads to an easier differentiation between ion signals and artefacts, and therefore easier data interpretation.     

Charge State Coalescence During Electrospray Ionization Improves Peptide Identification by Tandem Mass Spectrometry

We report the effects of supercharging reagents dimethyl sulphoxide (DMSO) and m-nitrobenzyl alcohol (m-NBA) applied to untargeted peptide identification, with special emphasis on non-tryptic peptides. Peptides generated from a mixture of five standard proteins digested with trypsin, elastase, or pepsin were separated with nanoflow liquid chromatography using mobile phases modified with either 5% DMSO or 0.1%m-NBA. Eluting peptides were ionized by online electrospray and sequenced by both CID and ETD using data-dependent MS/MS. Statistically significant improvements in peptide identifications were observed with DMSO co-solvent. In order to understand this observation, we assessed the effects of supercharging reagents on the chromatographic separation and the electrospray quality. The increase in identifications was not due to supercharging, which was greater for the 0.1%m-NBA co-solvent and not observed for the 5.0% DMSO co-solvent. The improved MS/MS efficiency using the DMSO modified mobile phase appeared to result from charge state coalescence.     

薄荷卷烟中香味成分的全二维气相色谱/飞行时间质谱分析

采用全二维气相色谱/飞行时间质谱法(GC×GC/TOFMS),以较长的非极性柱DB-5MS(30m×0.25mm×0.25μm)作为第一维柱,较短的中等极性柱DB-17MS(2m×0.1mm×0.1μm)作为第二维柱,利用固相微萃取法作为香味成分的萃取方法,对薄荷型ESSE卷烟的核心香味成分进行了定性分析,TOFMS谱图库检索结合全二维特有的包含结构信息的二维谱图,通过族分离和结构谱图鉴定,共鉴定了187种挥发性成分,其中对香气有贡献的成分118种.     

Papain Purification Insights: Monitoring by Electrophoretic Approaches and MALDI-TOF Peptide Mass Fingerprint Analyses

Papain was purified from dried Carica papaya latex by fractioned salt precipitation in presence of sodium tetrathionate to preserve enzymatic activity. Purification was followed by different electrophoretic methods. Identification of the purified product was afforded by submitting the peptides obtained by tryptic digestion of papain to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis. Comparison of the peptide masses analyzed by peptide mass fingerprinting (PMF) MALDI-TOF and those obtained by theoretical tryptic digestion, revealed the presence of some peptides belonging the other three endopeptidases contained in papaya latex (very similar to papain in molecular weight and pI) in the purified fraction of papain. The PMF by MALDI-TOF could be applied as a method to follow papain purification.     

A Two-Dimensional 1H NMR Approach for Peptide Backbone Assignments: Gramicidin S

Two-dimensional NMR spectra of gramicidin S have been measured. With the combination of COSY (through bond interaction) and NOESY (through space interaction) spectra, it is possible to identify amino acid residues sequentially. It is found that the phase-sensitive 2D technique is superior to the absolute value mode. A double quantum filtering technique could be combined with phase-sensitive COSY experiments to obtain the optimum results.     

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (b_n-1 + H₂O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H₂O and NH₃) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a y_m-1 ion, i.e., to the loss of the N-terminal residue following the a₁-y_m–1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.     

液相色谱-串联质谱法测定牛奶中5种多肤类抗生素

建立了牛奶中杆菌肽、粘杆菌素A、粘杆菌素B、维吉尼霉素和万占霉素5种多肽类抗生素的反相液相色谱-串联质谱(HPLC-MS/MS)检测方法.牛奶样品经甲醇-0.1%甲酸水提取后,用4%三氯乙酸乙睛除蛋白,液-液萃取后,采用0.1%甲酸(A)和0.1%甲酸乙腈(B)作为流动相进行梯度洗脱.质谱(ESI+)采用多离子检测模式...     

The Structure of the Diatom Silaffin Peptide R5 within Freestanding Two-Dimensional Biosilica Sheets

The silaffin peptide R5 is instrumental to the mineralization of silica cell walls of diatom organisms. The peptide is also widely employed in biotechnology, for example, in the encapsulation of enzymes and for fusion proteins in tissue regeneration. Despite its scientific and technological importance, the interfacial structure of R5 during silica precipitation remains poorly understood. We herein elucidate the conformation of the peptide in its active form within silica sheets by interface-specific vibrational spectroscopy in combination with molecular dynamics simulations. Contrary to previous solution-state NMR studies, our data confirm that R5 maintains a defined structure when interacting with extended silica sheets. We show that the entire amino acid sequence of R5 interacts with silica during silica formation, leading to the intercalation of silica into the assembled peptide film.     

Chimera Spectrum Diagnostics for Peptides Using Two-Dimensional Partial Covariance Mass Spectrometry

The rate of successful identification of peptide sequences by tandem mass spectrometry (MS/MS) is adversely affected by the common occurrence of co-isolation and co-fragmentation of two or more isobaric or isomeric parent ions. This results in so-called `chimera spectra’, which feature peaks of the fragment ions from more than a single precursor ion. The totality of the fragment ion peaks in chimera spectra cannot be assigned to a single peptide sequence, which contradicts a fundamental assumption of the standard automated MS/MS spectra analysis tools, such as protein database search engines. This calls for a diagnostic method able to identify chimera spectra to single out the cases where this assumption is not valid. Here, we demonstrate that, within the recently developed two-dimensional partial covariance mass spectrometry (2D-PC-MS), it is possible to reliably identify chimera spectra directly from the two-dimensional fragment ion spectrum, irrespective of whether the co-isolated peptide ions are isobaric up to a finite mass accuracy or isomeric. We introduce ‘3-57 chimera tag’ technique for chimera spectrum diagnostics based on 2D-PC-MS and perform numerical simulations to examine its efficiency. We experimentally demonstrate the detection of a mixture of two isomeric parent ions, even under conditions when one isomeric peptide is at one five-hundredth of the molar concentration of the second isomer.     

全二维气相色谱/飞行时间质谱用于烟用香精化学组分的分析

采用全二维气相色谱/飞行时间质谱(GC×GC/TOFMS),以较长的极性柱DB-WAX(60 m×0.25 mm×0.25μm)作为第一柱,较短的中等极性柱DB-1701(3 m×0.1 mm,0.4μm)作为第二柱,对一种国产香烟中常用的烟用香精进行了分析,共鉴定出相似度和反相似度分别大于800和900的化合物有84种(在相同条件下,GC/MS鉴定出的组分数仅为21种)。在与GC×GC/TOFMS相同的分析条件下,利用多内标定量法,用GC-GC/氢火焰离子化检测器(FID)进一步对这些组分进行了准确定量。实验结果很好地解释了该香精的致香机理。     

全二维气相色谱/飞行时间质谱用于柴油组成的研究

将全二维气相色谱法(GC×GC)用于柴油馏分的组成分布研究,建立了两种GC×GC方法,一种用于柴油组成的详细表征,另一种用于柴油族组成的快速分离和定量,两种方法均不需要样品预处理。用前一种方法对柴油馏分中的烃类化合物、主要的含硫化合物与含氮化合物组成进行了研究;对催化裂解柴油中的27种含氮化合物和42种含硫化合物进行了定性;用后一种方法在70 min内即可完成柴油馏分族组成的定量分析,应用所建立的方法测定了4个不同来源的柴油馏分中非芳烃、一环芳烃、二环芳烃、三环芳烃的含量,定量结果与ASTM D2425法