The logic of protein post-translational modifications (PTMs): Chemistry,mechanisms and evolution of protein regulation through covalent attachments

Protein post-translational modifications (PTMs) play a crucial role in all cellular functions by regulating protein activity, interactions and half-life. Despite the enormous diversity of modifications, various PTM systems show parallels in their chemical and catalytic underpinnings. Here, focussing on modifications that involve the addition of new elements to amino-acid sidechains, I describe historical milestones and fundamental concepts that support the current understanding of PTMs. The historical survey covers selected key research programmes, including the study of protein phosphorylation as a regulatory switch, protein ubiquitylation as a degradation signal and histone modifications as a functional code. The contribution of crucial techniques for studying PTMs is also discussed. The central part of the essay explores shared chemical principles and catalytic strategies observed across diverse PTM systems, together with mechanisms of substrate selection, the reversibility of PTMs by erasers and the recognition of PTMs by reader domains. Similarities in the basic chemical mechanism are highlighted and their implications are discussed. The final part is dedicated to the evolutionary trajectories of PTM systems, beginning with their possible emergence in the context of rivalry in the prokaryotic world. Together, the essay provides a unified perspective on the diverse world of major protein modifications.     

JUMPptm: Integrated software for sensitive identification of post-translational modifications and its application in Alzheimer's disease study

Background : Mass spectrometry (MS)-based proteomic analysis of posttranslational modifications (PTMs) usually requires the pre-enrichment of modified proteins or peptides. However, recent ultra-deep whole proteome profiling generates millions of spectra in a single experiment, leaving many unassigned spectra, some of which may be derived from PTM peptides. Methods : Here we present JUMPptm, an integrative computational pipeline, to extract PTMs from unenriched whole proteome. JUMPptm combines the advantages of JUMP, MSFragger and Comet search engines, and includes de novo tags, customized database search and peptide filtering, which iteratively analyzes each PTM by a multi-stage strategy to improve sensitivity and specificity. Results : We applied JUMPptm to the deep brain proteome of Alzheimer's disease (AD), and identified 34,954 unique peptides with phosphorylation, methylation, acetylation, ubiquitination, and others. The phosphorylated peptides were validated by enriched phosphoproteome from the same sample. TMT-based quantification revealed 482 PTM peptides dysregulated at different stages during AD progression. For example, the acetylation of numerous mitochondrial proteins is significantly decreased in AD. A total of 60 PTM sites are found in the pan-PTM map of the Tau protein. Conclusion : The JUMPptm program is an effective tool for pan-PTM analysis and the resulting AD pan-PTM profile serves as a valuable resource for AD research.     

一种联合MALDI-TOF MS直接鉴定阳性血培养瓶方法改进及与Sepsityper Kit试剂盒法、SELTERS法和血清分离胶法的比较

目的评价直接使用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)联合Sepsityper Kit试剂盒法(简称试剂盒法)、SELTERS法和血清分离胶法(简称分离胶法)鉴定阳性血培养瓶血中细菌的符合率,并对SELTERS方法进行改进,以缩短样本处理时间。方法对656例临床血培养阳性标本,应用试剂盒法、SELTERS方法或分离胶法处理后,直接使用质谱仪快速鉴定菌株,同时进行传统培养,比较分析二者之间的差异。结果656例血培养阳性标本共分离出626株单种菌感染和30株多种菌感染标本。MALDI-TOF MS联合试剂盒法、SELTERS法或分离胶法可在1 h内快速鉴定血培养阳性标本。在单种细菌感染中,MALDI-TOF MS联合试剂盒法直接鉴定革兰阳性菌的种、属符合率分别是66.8%(141/211)、21.3%(45/211),革兰阴性菌的种、属符合率分别是97.1%(367/378)、0.8%(3/378),真菌的种、属符合率分别是32.4%(12/37)、0.0%(0/37);MALDI-TOF MS联合SELTERS法直接鉴定革兰阳性菌的种、属符合率分别是66.8%(141/211)、21.3%(45/211),革兰阴性菌的种、属符合率分别是96.3%(364/378)、2.4%(9/378),真菌的种、属符合率分别是32.4%(12/37)、2.7%(1/37);MALDI-TOF MS联合分离胶法直接鉴定革兰阳性菌的种、属符合率分别是51.2%(108/211)、20.9%(44/211),革兰阴性菌的种、属符合率分别是93.4%(353/378)、1.6%(6/378),真菌的种、属符合率分别是13.5%(5/37)、2.7%(1/37);MALDI-TOF MS联合改良SELTERS法直接鉴定革兰阳性菌的种、属符合率分别是59.1%(13/22)、18.2%(4/22),革兰阴性菌的种、属符合率分别是88.5%(23/26)、3.8%(1/26),真菌的种、属符合率分别是0.0%(0/2)、50.0%(1/2)。而对于多种菌感染的血培养瓶,3种方法鉴定率均较低。结论MALDI-TOF MS联合试剂盒法、SELTERS法或分离胶直接鉴定阳性血标本中的病原菌,其结果可在1 h内获得,并与传统培养结果相比具有较高的符合率。但是这些方法检测更快速、操作更简便,同时改良SELTERS法样本处理时间缩短,成本降低,且符合率与前3种方法没有区别。这4种方法均能满足临床快速诊断和及时有效抗菌治疗的需求,临床可根据自身情况选择。     

Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0–592.5 μg/m³). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n = 73) was highly significant (p < 0.0001, Student's t-test) for both HPLC/MS/MS methods (r = 0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI^® documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.     

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis)

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35 kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35 kDa protein was ~ 98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.     

Enzymatic and radioimmunoassay procedures combined with electrophoresis and HPLC for the recovery and characterization of substance P in human brain.

Immunoreactive substance P was recovered from human brain (hypothalamus and substantia nigra) by acetic acid extraction, ion exchange chromatography (SP-Sephadex), molecular sieving (Sephadex G-50) and column electrophoresis in agarose suspension. The chemical nature of the active material was further studied with various biochemical techniques including agarose suspension electrophoresis, HPLC and different kinds of enzyme radioimmunoassays. By combining these techniques it was possible to confirm structure identity between the recovered active component and substance P previously isolated from bovine brain. Thus, the major activity reacting with the substance P antibodies was indistinguishable from the synthetic bovine analogue in all chromatographic systems including analytical electrophoresis at different pH:s and HPLC. Furthermore, digestion of the active material with post-proline cleaving enzyme and trypsin yielded fragments identical with those expected from the bovine peptide as confirmed by specific radioimmunoassays in conjunction with electrophoresis or HPLC. The result also indicates the usefulness of the present procedures for identifying peptides structures available only in minute amounts.     

微孔草籽油的超临界CO2流体萃取及HPLC—MS分析

利用超临界CO2萃取微孔草籽油,并对籽油进行了HPLC／MS分析。实验确定的最佳超临界CO2流体萃取条件是：萃取温度45℃,萃取压力20MPa,CO2流量为35-40kg／h,萃取时间120min,在此条件下白刺籽油的萃取率为16．12％。利用HPLC／MS对微孔草籽油分析,发现其不饱和脂肪酸的相对含量高达73．19％。比较了超临界CO2萃取微孔草籽油油样和石油醚萃取微孔草籽油油样的理化性质,发现超临界CO2流体萃取的籽油质量优于传统溶剂萃取的籽油。     

HPLC fingerprints combined with principal component analysis,hierarchical cluster analysis and linear discriminant analysis for the classification and differentiation of Peganum sp. indigenous to China

Introduction – Seeds of wild Peganum harmala Linn., P. multisectum (Maxim) Bobr., P. nigellastrum Bunge and a probable indeterminate species, herein referred to as P. variety, are commonly used in Chinese medicine. These seeds cannot be differentiated based on morphology. Objective – Seeds of P. harmala Linn., P. multisectum (Maxim) Bobr., P. nigellastrum Bunge and P. variety were collected in different provinces in China and their HPLC profiles were recorded for statistical analysis and pattern recognition. Methodology – HPLC chromatograms of seed extracts were recorded under the same conditions. Individual HPLC chromatograms for each species were evaluated against the mean chromatogram for the same species generated using a similarity evaluation computer program. Data from chromatographic fingerprints were also processed using principal component analysis (PCA), hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA). Results – The Peganum sp. seed extracts had similar HPLC fingerprints but with some inter‐specific differences. The chromatographic fingerprints combined with PCA, HCA and LDA could distinguish the seeds of the different species of Peganum investigated. Conclusion – HPLC fingerprints can be used to authenticate and differentiate the seeds of three different species of genus Peganum indigenous to China. The results indicated that the unidentified P. variety might indeed be a new species or variety. Copyright © 2009 John Wiley & Sons, Ltd.     

Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor.

Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.     

The primary structure of Bacillus subtilis acidic ribonsomal protein B-19. Isolation and characterization of peptides and the complete amino acid sequence

The complete primary structure of Bacillus subtilis acidic protein B-L9, functionally equivalent to protein L7/L12 from E. coli, has been determined. B-L9 is composed of 122 residues and has the amino acid composition: Asp3, ASN3, Thr4, Ser3, Glu22, Gln1, Pro3, Gly11, Ala21, Val14, Ile9, Leu12, Phe2, Lys13, and Arg1. The molecular weight of B-L9 is 12,633. The amino acid sequence was determined by a combination of automated Edman degradation of the intact protein in a modified Beckman sequenator, and micro dansyl-Edman degradation of the peptides obtained from digestions with trypsin, thermolysin, Staphylococcus aureus protease, chymotrypsin and pepsin. A comparison of protein B-L9 from B. subtilis with E-L12 from E. coli shows a relatively high degree of homology.     

Influenza A virus protein PB1-F2: synthesis and characterization of the biologically active full length protein and related peptides.

Recently the discovery of a novel 87 amino acid influenza A virus (IAV) protein, named PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most of the known human IAV isolates. Using optimized protocols, full length biologically active sPB1-F2 and a number of fragments have been synthesized by following either the standard elongation SPPS method or by native chemical ligation of unprotected N- and C-terminal peptide fragments at the histidine and cysteine residues located in position 41 and 42 of the native sequence, respectively. The ligation procedure afforded the most efficient synthesis of sPB1-F2 and facilitated the generation of various mutants of sPB1-F2 from pre-synthesized peptide fragments. During the synthesis of sPB1-F2, the formation of succinimide and subsequent conversion to the piperidine derivative at the aspartic acid residue in position 23 was observed. This reaction was forestalled by applying specific modifications to the SPPS protocol. The chain-elongation SPPS protocol is optimal for producing small peptides of sPB1-F2, their derivatives and precursors for a subsequent ligation protocol, while the full length protein, mutants and labelled derivatives are more conveniently and efficiently synthesized by SPPS protocols that include native chemical ligation. The molecular identity of sPB1-F2 was confirmed by peptide mapping, mass spectrometry, N-terminal sequencing, (1)H NMR spectroscopy and Western blot analysis. The latter analysis afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization, a phenomenon observed both for full length sPB1-F2 and fragments thereof, as well as for its full length viral counterpart. Our synthesis protocols open the field for multiple biological and structural studies on sPB1-F2 that, similar to the molecule expressed in an IAV context, induces apoptosis and interacts with membranes in vitro and in vivo, as shown in previous studies.     

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.     

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.     

Analytical biotechnology of recombinant peptides and proteins: II. A confirmation of the primary structure of fusion protein containing human proinsulin and optimization of its proteolysis by trypsin

The kinetics of trypsin proteolysis of the fusion protein (FP) containing human proinsulin was studied by a set of analytical micromethods. These were the microcolumn reversed-phase HPLC and the qualitative identification by MALDI-TOF mass spectrometry and amino acid sequencing. The first stage of the proteolysis was shown to be the cleavage of FP into the leader fragment and proinsulin. The subsequent splitting off ofC-peptide from proinsulin results in the formation of Arg^B31-Arg^B32-insulin. The effect of temperature on the formation of de-Thr^B30-insulin, a by-product, was also studied. The structure of FP was confirmed by the peptide mapping technique, and the leader fragment was shown to contain noN-terminal Met residue. For communication I, see [1].     

Isolation and characterization of precursors in bacteriophage T4 baseplate assembly. III. The carboxyl termini of protein P11 are required for assembly activity

The assembly activity and electrophoretic mobility of a T4 bacteriophage baseplate protein, P11, have been found to be affected by digestion with the proteases trypsin, subtilisin and carboxypeptidase Y. Analysis of the trypsin limit-digestion product of P11 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and size analysis by high performance liquid chromatography indicate that there is a decrease of approximately 5000 in the molecular weight of the P11 molecule or a loss of 2500 in Mr from each of the gp11 subunits of the dimer. During protease treatment P11 demonstrates a time-dependent loss in the ability to interact with the baseplate protein P10 to form the P(10/11) complex, the first assembly intermediate of the T4 baseplate 1/6th arm. Similar treatments of the P(10/11) complex indicate that P11 in the complex is not affected by these proteases. Concomitant with the loss of assembly activity is a change in the electrophoretic mobility of P11 on non-denaturing polyacrylamide gels from a single band to a series of more mobile bands suggesting sequential loss of positive charge. P11 assembly activity is completely lost after removal of the first positive charge. These results suggest that the carboxyl termini of the two gp11 subunits of the P11 molecule are involved in the interaction of P11 with P10 to form the P(10/11) complex. Analysis of the portion of gp11 removed by carboxypeptidase Y demonstrates that there are up to 13 aliphatic and aromatic carboxyl-terminal amino acids.     

Purification, characterization, and primary structure of Escherichia coli protease VII with specificity for paired basic residues: identity of protease VII and OmpT.

Escherichia coli cells were found to contain a novel outer membrane-associated protease, designated protease VII (K. Sugimura and N. Higashi, J. Bacteriol. 170:3650-3654, 1988). This enzyme was purified to homogeneity and exhibited an apparent molecular weight of 36,000 on sodium dodecyl sulfate gels and 180,000 on a TSK G-3000SW column in the presence of Triton X-100. It was capable of cleaving several peptides at the center of paired basic residues but not at single basic residues, implying that it is distinct from trypsinlike proteases. Protease VII was most active at pH 6.0 and was sensitive to a serine protease inhibitor, diisopropylfluorophosphate, and to the bivalent cations Zn2+, Cu2+, and Fe2+. The nucleotide sequence of a protease VII gene-carrying DNA fragment, which had been cloned by complementation analysis (K. Sugimura, Biochem. Biophys. Res. Commun. 153:753-759, 1988) was determined. It carried two putative promoter regions and a putative Shine-Dalgarno sequence in addition to the complete structural gene, which encoded pre-protease VII of 317 amino acid residues, with the N-terminal 20 residues being a signal peptide. By comparing their amino acid sequences, protease VII and OmpT, which specifically cleaves ferric enterobactin receptor protein, were found to be identical.     

A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization

In the present studies, we have purified a novel small Mr GTP-binding protein, designated as smg p21, to near homogeneity from bovine brain crude membranes, isolated the complementary DNA (cDNA) of this protein from a bovine brain cDNA library, determined the complete nucleotide and deduced amino acid sequences, and characterized the kinetic properties. The cDNA of smg p21 has an open reading frame encoding a protein of 184 amino acids with a calculated Mr of 20,987. The Mr of purified smg p21 is estimated to be about 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Homology search indicates that smg p21 is a novel protein with the consensus amino acid sequences for GTP/GDP-binding and GTPase domains but shares about 55% amino acid sequence homology with the human c-Ha-ras protein. Moreover, smg p21 has the same putative effector domain as the Ha-, Ki-, and N-ras proteins at the same position and the same consensus C-terminal sequence as in these ras proteins. Consistent with these structural properties, smg p21 binds specifically [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), GTP, and GDP with a Kd value for GTP gamma S of about 40 nM. smg p21 binds about 0.7 mol of GTP gamma S/mol of protein. [35S]GTP gamma S-binding to smg p21 is inhibited by pretreatment with N-ethylmaleimide.smg p21 hydrolyzes GTP to liberate Pi with a turnover number of about 0.007 min-1. These kinetic properties of smg p21 are similar to those of the c-ras proteins. These results suggest that smg p21 is a novel GTP-binding protein exerting action(s) similar or antagonistic to that (those) of the ras proteins.     

Structural requirements for localization and activation of protein kinase C mu (PKC mu) at the Golgi compartment.

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC) mu involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKC mu-green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the NH(2)-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKC mu completely abrogated Golgi localization of PKC mu. As an NH(2)-terminal PKC mu fragment was colocalized with p24, this region of PKC mu is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKC mu to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKC mu found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKC mu, in which Golgi compartment recruitment precedes and is essential for activation loop phosphorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH(2)-terminal serine(s) in the regulatory domain. PKC mu activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKC mu function at the Golgi compartment.