A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis

Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.     

A Simple and Gentle Method for Concentrating Protein Solutions

A gentle method for concentrating very dilute protein solutions is described. The high capacity of aminohexylagarose in adsorbing different proteins is utilized to handle small or large amounts of protein with practically no losses in material or activity. To concentrate very dilute protein solutions as they occur during purification procedures e. g. of enzymes, a one-step non-inactivating nethod is needed that may easily be integrated into the purification programm.

In the course of the purification of a labile enzyme (1) we developed a simple chromatographic method which seems to work for a large variety of proteins. The procedure is applicable to very dilute protein solutions, to small samples as well as to large scale preparations, and it is relatively inexpensive. It appears to be a very gentle method since in all cases tested no loss of enzymic activity could be observed.     

A Rapid Method For Concentrating Small Organisms For Sectioning

Lipp, Walther. Histochemische Methoden. About 24 pages per issue; 3 issues (No. 9, 10 and 11) during 1956. 16 £ 23 cm. R. Oldenbourg, München 1, Germany. DM 30.—for 6 issues; DM 6.—per single copy. (In German)

Molenaar, I. Ontkalking van harde weefsals. Een histochemische studie. Thesis, Rijksuniversiteit te Utrecht, 1957. pp. 124.     

A Proteomic Strategy for Global Analysis of Plant Protein Complexes

Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.     

Protein Arginylation in Rat Brain Cytosol: A Proteomic Analysis

Arginine can be post-translationally incorporated from arginyl-tRNA into the N-terminus of soluble acceptor proteins in a reaction catalyzed by arginyl-tRNA protein transferase. In the present study, several soluble rat brain proteins that accepted arginine were identified after arginine incorporation by two dimensional electrophoresis and mass spectrometry. They were identified as: contrapsin-like protease inhibitor-3, α-1-antitrypsin, apolipoprotein E, hemopexin, calreticulin and apolipoprotein A-I. All of these proteins shared a signal sequence for the translocation of proteins across endoplasmic reticulum membranes. After losing the signal peptide, these proteins expose amino acids described as compatible for post-translational arginylation. Although the enzymatic system involved in arginylation is confined mainly in cytosol and nucleus, all the substrates described herein enter to the exocytic pathway co-translationally. Therefore, we postulate that the substrates for arginylation could reach the cytosol by retro-translocation and be then arginylated.     

Interaction Network of Proteins Associated with Human Cytomegalovirus IE2-p86 Protein during Infection: A Proteomic Analysis

Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection. The protein profile at 8, 24, and 48 h post infection revealed that UL84 tightly associated with IE2-p86, and more viral and cellular proteins came into association with IE2-p86 with the progression of virus infection. A computational analysis of the protein-protein interaction network indicated that all of the 9 viral proteins and most of the cellular proteins identified in the study are interconnected to varying degrees. Of the cellular proteins that were confirmed to associate with IE2-p86 by immunoprecipitation, C1QBP was further shown to be upregulated by HCMV infection and colocalized with IE2-p86, UL84 and UL44 in the virus replication compartment of the nucleus. The IE2-p86 interactome network demonstrated the temporal development of stable and abundant protein complexes that associate with IE2-p86 and provided a framework to benefit future studies of various protein complexes during HCMV infection.     

A Rapid Method for Purification of Myelin Basic Protein

A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.     

A Rapid Method for Protein Extraction from Fission Yeast

Researchers working with fission yeast conduct protein extraction widely and frequently, but this includes the handling of glass beads, and hence is laborious and cumbersome, especially when dealing with a large number of samples. Here we describe a rapid and reliable method for preparing protein extract from fission yeast, one which is applicable to routine western blotting.     

Proteomic Screening for Amyloid Proteins

Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.     

SIMS: A Hybrid Method for Rapid Conformational Analysis

Proteins are at the root of many biological functions, often performing complex tasks as the result of large changes in their structure. Describing the exact details of these conformational changes, however, remains a central challenge for computational biology due the enormous computational requirements of the problem. This has engendered the development of a rich variety of useful methods designed to answer specific questions at different levels of spatial, temporal, and energetic resolution. These methods fall largely into two classes: physically accurate, but computationally demanding methods and fast, approximate methods. We introduce here a new hybrid modeling tool, the Structured Intuitive Move Selector (sims), designed to bridge the divide between these two classes, while allowing the benefits of both to be seamlessly integrated into a single framework. This is achieved by applying a modern motion planning algorithm, borrowed from the field of robotics, in tandem with a well-established protein modeling library. sims can combine precise energy calculations with approximate or specialized conformational sampling routines to produce rapid, yet accurate, analysis of the large-scale conformational variability of protein systems. Several key advancements are shown, including the abstract use of generically defined moves (conformational sampling methods) and an expansive probabilistic conformational exploration. We present three example problems that sims is applied to and demonstrate a rapid solution for each. These include the automatic determination of “active” residues for the hinge-based system Cyanovirin-N, exploring conformational changes involving long-range coordinated motion between non-sequential residues in Ribose-Binding Protein, and the rapid discovery of a transient conformational state of Maltose-Binding Protein, previously only determined by Molecular Dynamics. For all cases we provide energetic validations using well-established energy fields, demonstrating this framework as a fast and accurate tool for the analysis of a wide range of protein flexibility problems.     

A Rapid Method for Characterization of Protein Relatedness Using Feature Vectors

We propose a feature vector approach to characterize the variation in large data sets of biological sequences. Each candidate sequence produces a single feature vector constructed with the number and location of amino acids or nucleic acids in the sequence. The feature vector characterizes the distance between the actual sequence and a model of a theoretical sequence based on the binomial and uniform distributions. This method is distinctive in that it does not rely on sequence alignment for determining protein relatedness, allowing the user to visualize the relationships within a set of proteins without making a priori assumptions about those proteins. We apply our method to two large families of proteins: protein kinase C, and globins, including hemoglobins and myoglobins. We interpret the high-dimensional feature vectors using principal components analysis and agglomerative hierarchical clustering. We find that the feature vector retains much of the information about the original sequence. By using principal component analysis to extract information from collections of feature vectors, we are able to quickly identify the nature of variation in a collection of proteins. Where collections are phylogenetically or functionally related, this is easily detected. Hierarchical agglomerative clustering provides a means of constructing cladograms from the feature vector output.     

A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

运用蛋白质组学技术研究与氨甲蝶呤耐药性相关的蛋白质

氨甲蝶呤（MTX）是一种重要的常用化疗药物,然而由于肿瘤细胞对其耐药性的增强而经常导致其疗效大大降低。为了寻找并鉴定与MTX耐药性相关的蛋白质从而为进一步闸明MTX的耐药机制提供线索,培养来源于小鼠NIH3T3的小鼠胚胎成纤维细胞系3T3R500与其耐300μmol／L MTX的细胞系MTX300,提取上述两种细胞系的总蛋白质,双向凝胶电泳分离蛋白质组成分,扫描并通过软件分析考马斯亮蓝染色的2-DE凝胶,选取表达差异最显著的点,胶内酶切后MALDI-TOF-MS进行肽指纹图谱（PMF）鉴定。图像分析显示,实验组和对照组的蛋白质组图谱之间,一些蛋白质点的表达有明显的变化。通过MALDI-TOF-MS和数据库查询,成功鉴定了耐药后表达变化最显著的蛋白质点为二氢叶酸还原酶（DHFR）,并通过Western blot验证了该结果,提示DHFR在MTX耐药机制中发挥重要作用。     

Proteomic Analysis of Neorickettsia sennetsu Surface-Exposed Proteins and Porin Activity of the Major Surface Protein P51

Neorickettsia sennetsu is an obligate intracellular bacterium of monocytes and macrophages and is the etiologic agent of human Sennetsu neorickettsiosis. Neorickettsia proteins expressed in mammalian host cells, including the surface proteins of Neorickettsia spp., have not been defined. In this paper, we isolated surface-exposed proteins from N. sennetsu by biotin surface labeling followed by streptavidin-affinity chromatography. Forty-two of the total of 936 (4.5%) N. sennetsu open reading frames (ORFs) were detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS), including six hypothetical proteins. Among the major proteins identified were the two major β-barrel proteins: the 51-kDa antigen (P51) and Neorickettsia surface protein 3 (Nsp3). Immunofluorescence labeling not only confirmed surface exposure of these proteins but also showed rosary-like circumferential labeling with anti-P51 for the majority of bacteria and polar to diffuse punctate labeling with anti-Nsp3 for a minority of bacteria. We found that the isolated outer membrane of N. sennetsu had porin activity, as measured by a proteoliposome swelling assay. This activity allowed the diffusion of l-glutamine, the monosaccharides arabinose and glucose, and the tetrasaccharide stachyose, which could be inhibited with anti-P51 antibody. We purified native P51 and Nsp3 under nondenaturing conditions. When reconstituted into proteoliposomes, purified P51, but not Nsp3, exhibited prominent porin activity. This the first proteomic study of a Neorickettsia sp. showing new sets of proteins evolved as major surface proteins for Neorickettsia and the first identification of a porin for the genus Neorickettsia.Neorickettsia spp. are unique environmental, Gram-negative, obligate intracellular bacteria maintained in nature through vertical transmission in trematodes (16, 17, 39, 42). Neorickettsia spp. are polymorphic cocci belonging to the family Anaplasmataceae within the order Rickettsiales in the class Alphaproteobacteria (7). Neorickettsia sennetsu (formerly called Rickettsia sennetsu or Ehrlichia sennetsu) is the first human pathogen in the family Anaplasmataceae to have been isolated and cultured (11, 34). N. sennetsu infects human monocytes and macrophages and causes the disease Sennetsu neorickettsiosis (10, 34, 41). Epidemiologic studies of Sennetsu neorickettsiosis show a strong link between the human ingestion of metacercaria-infested gray mullet fish and acquisition of the disease (11). Symptoms are similar to those of infectious mononucleosis and include swelling of the lymph nodes, pyrexia, inappetence, lethargy, sleeplessness, and overall malaise (10, 34, 41). Geographically, N. sennetsu infections have been reported mainly in western and southern Japan, although antibodies to N. sennetsu have also been found in humans in Malaysia, and one strain of N. sennetsu has been isolated from Malaysia (10, 19, 44). Recently, N. sennetsu infection was found in Laos (35). Treatment of Sennetsu neorickettsiosis involves tetracycline therapy and is normally highly successful at resolving the symptoms (10).Gram-negative bacteria generally have porins spanning their outer membranes. These proteins enable the transport of hydrophilic molecules, such as amino acids, sugars, and other nutrients (36). As seen in other members of the Anaplasmataceae, N. sennetsu is limited in its ability to synthesize necessary compounds, including amino acids and enzymes for intermediary metabolism and glycolysis (20). Therefore, porins are an absolute necessity for the survival of this bacterium. To date, the only porins defined for the order Rickettsiales are major outer membrane proteins of Anaplasma phagocytophilum named P44s (22) and OMP-1F and P28 in Ehrlichia chaffeensis (26). These porins contain 16 (P44s) or 12 (OMP-1F and P28) transmembrane passes, and some are large enough to allow the slow diffusion of tetrasaccharides. P44/Msp2 and OMP-1/P28/P30 proteins belong to the family pfam01617, and the N. sennetsu genome was reported to encode only one hypothetical protein from this family (GenBank accession no. NSE_0875) (20), later named Neorickettsia surface protein 3 (Nsp3) (29).In the present study, surface-exposed proteins of N. sennetsu were isolated from cell culture and identified by proteomics. We first isolated the outer membrane fraction from host cell-free N. sennetsu and examined porin activity by using an in vitro proteoliposome swelling assay. Second, we used antibodies against the dominant protein P51 to examine neutralization of the porin activity. Third, we purified native P51 and Nsp3 from the isolated outer membrane fraction by high-pressure liquid chromatography (HPLC) and tested whether these proteins have porin activity. Identification of surface-exposed proteins and the protein with major porin activity will help in understanding N. sennetsu and the disease that it causes.     

A Rapid HPLC Method to Separate the Triplet Proteins of Neurofilament

In this article a fast HPLC technique to separate the individual neurofilament proteins is described. Highly pure fractions of the three neurofilament proteins can be obtained. As much as 50 mg of each neurofilament polypeptide can be separated from a crude neurofilament protein preparation in one step in less than 2 h. The short separation time is of importance in minimizing degradation, especially of the 150-kilodalton neurofilament polypeptide.     

Proteomic Analysis of Osmotic Stress-Responsive Proteins in Sugarcane Leaves

Osmotic stress-related proteins in sugarcane were identified using proteomics approach based on two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Sugarcane settlings were subjected to osmotic stress in the nutrient solution containing 10% (w/v) PEG 6000 for 14 h. Total proteins were extracted from leaves, and separated by 2-DE. Four typical spots exhibited significant changes in PEG treatment compared to control, which were identified using MALDI-TOF-MS successfully. The drought inducible 22 kDa protein and Rubisco small subunit were up-regulated while isoflavone reductase-like (IRLs, related to antioxidant defense system) protein and delta chain of ATP synthase were down-regulated by the osmotic stress. Analysis of the results showed that the most differential proteins under osmotic stress were acidic, unstable and transmembrane proteins, enriched with hydrophobic amino acids such as leucine and alanine which are extremely important for structural stabilization of proteins by hydrophobic interaction. However, the drought inducible 22 kDa protein was a hydrophile and non-transmembrane protein enriched with glutamic acid. These results provide new insight into the part of regulatory mechanism of adaptations to osmotic stress through differential expression of specific proteins and implicate several previously unrecognized proteins to osmotic stress.     

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pI 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function.     

Web-Based Software for Rapid Top-Down Proteomic Identification of Protein Biomarkers,with Implications for Bacterial Identification

We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption ionization (MALDI), mass isolated, and fragmented using a tandem time of flight (TOF-TOF) mass spectrometer. The sequence-specific fragment ions generated were compared to a database of in silico fragment ions derived from bacterial protein sequences whose molecular weights are the same as the nominal molecular weights of the protein biomarkers. A simple peak-matching and scoring algorithm was developed to compare tandem mass spectrometry (MS-MS) fragment ions to in silico fragment ions. In addition, a probability-based significance-testing algorithm (P value), developed previously by other researchers, was incorporated into the software for the purpose of comparison. The speed and accuracy of the software were tested by identification of 10 protein biomarkers from three Campylobacter strains that had been identified previously by bottom-up proteomics techniques. Protein biomarkers were identified using (i) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions with all possible in silico N and C terminus fragment ions (i.e., ions a, b, b-18, y, y-17, and y-18), (ii) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions to residue-specific in silico fragment ions (i.e., in silico fragment ions resulting from polypeptide backbone fragmentation adjacent to specific residues [aspartic acid, glutamic acid, proline, etc.]), and (iii) fragment ion error analysis, which distinguished the systematic fragment ion error of a correct identification (caused by calibration drift of the second TOF mass analyzer) from the random fragment ion error of an incorrect identification.Food-borne illness is a serious and continuing problem, with an estimated 76 million cases in the United States per year (http://www.cdc.gov). It is often caused by bacteria and viruses that are often ubiquitous in the environment and are difficult to eliminate due to their ability to adapt. In addition to the resulting morbidity, food-borne illness also has enormous societal costs, including losses in worker productivity due to illness, recall of food products determined (or suspected) to be contaminated, etc. Consequently, there is a critical need to develop rapid and sensitive methods for detection and accurate identification of food-borne pathogens.A number of techniques have been developed for detection and identification of food-borne pathogens. A relatively recent technique for bacterial identification involves the use of mass spectrometry (MS). Because of its sensitivity and high specificity, MS has become a popular technique for chemicotaxonomic classification of microorganisms (16, 27). The use of MS in the analysis of microorganisms is a relatively recent application that was dramatically accelerated by the development of two ionization techniques in the late 1980s and early 1990s: electrospray ionization (15) and matrix-assisted laser desorption ionization (MALDI) (24, 37). When coupled with time of flight (TOF) MS, MALDI has been demonstrated to be a powerful tool for “fingerprinting” microorganisms by ionization and detection of proteins from intact bacterial cells or extracts resulting from bacterial cell lysis (1, 2, 3, 8-12, 19, 21, 25, 26, 29, 34, 40, 41, 42). Typically, MALDI-TOF MS “fingerprinting” of microorganisms involves analysis using either pattern recognition or bioinformatic algorithms.Pattern recognition analysis compares MALDI-TOF MS spectra of samples of unknown microorganisms to spectra of known microorganisms. A high degree of similarity between the MS spectrum of an unknown microorganism and an MS spectrum of a known microorganism strongly suggests the identity of the unknown microorganism (22, 39, 43). It should be noted that pattern recognition analysis does not rely on actual identification of the biomarker ion peaks in an MS spectrum. It is the pattern generated by multiple ion peaks that constitutes a microorganism''s “fingerprint.” The actual identities of individual ion peaks are not specified, and the peaks could be peaks for any of a number of possible biological molecules generated by a microorganism, including proteins, nucleic acids, lipids, etc.Microorganism identification by bioinformatic analysis of MALDI-TOF MS data involves using the protein molecular weights (MWs) in bacterial genomic databases to assign biomarker ion peaks in a mass spectrum to specific proteins (4, 5, 32, 33, 45). If a significant number of biomarker ion peaks in a mass spectrum correspond to protein MWs for the open reading frames of a microorganism''s genome, then the microorganism is considered identified. Such an analysis has also incorporated the simplest and most common posttranslational modification (PTM) observed for bacterial proteins, N-terminal methionine cleavage (5). It should be noted, however, that “identification” of a microorganism relies solely on a sufficient number of protein MWs derived from open reading frames of its genome corresponding to the m/z of biomarker ions in a MALDI-TOF MS spectrum. However, the protein MW alone is not sufficient to definitively identify a biomarker ion as a specific protein. Protein biomarkers are considered to be tentatively assigned instead of definitively identified.Analysis of samples containing multiple bacterial organisms presents increased challenges for MALDI-TOF MS when protein MW is the sole criterion for protein biomarker identification. Clearly, it would be advantageous if researchers could obtain more information about a biomarker in addition to its MW. In the case of protein biomarkers, this can be accomplished by enzymatically digesting a protein in solution and analyzing its tryptic peptides by MS (peptide mass mapping) or by tandem MS (MS-MS) (sequence tags) (45). Alternatively, it is possible to fragment mature, intact proteins (without digestion) in the gas phase to obtain sequence-specific and PTM information. This approach is referred to as top-down proteomics. Until recently, top-down proteomics was possible only if Fourier transform ion cyclotron resonance MS involving complicated gas phase ion dissociation techniques was used (6, 23).Although not originally designed for top-down proteomics, recently developed MALDI-tandem TOF (MALDI-TOF-TOF) MS was shown to fragment small or modest-size proteins (5 kDa > molecular mass < 15 kDa) without prior digestion (28). Demirev and coworkers (7) identified Bacillus atrophaeus and Bacillus cereus spores by fragmenting their protein biomarkers using a MALDI tandem mass spectrometer and analyzing the sequence-specific fragment ions generated by comparison to in silico fragment ions derived from protein amino acid sequences from genomic databases. Protein and microorganism identities were determined using a probability-based significance-testing algorithm (P value). The P value algorithm calculates the probability that a protein or microorganism identification occurred randomly. The smaller the P value, the lower the probability that an identification occurred randomly. The data analysis was performed using software developed in house (7).In the current study, web-based software and databases, developed in house at the U.S. Department of Agriculture (USDA), were used to identify 10 protein biomarkers from three pure strains of Campylobacter by sequence-specific fragmentation using a MALDI-TOF-TOF mass spectrometer. Many of the protein biomarkers had been identified previously by bottom-up proteomics techniques (9, 11, 12), which provided an excellent data set to test the accuracy and performance of the algorithms incorporated into the software. MALDI-TOF-TOF MS-MS fragment ions were compared with a database of in silico fragment ions derived from bacterial protein sequences. The sequence-specific MS-MS fragment ions were used to identify a protein and thus the source microorganism. A simple peak-matching mathematical algorithm, incorporated into the software, was used to score and rank protein and microorganism identifications. In addition, the P value algorithm of Demirev and coworkers (7) was also incorporated into the USDA software (available with execution of appropriate control usage agreement) for comparison to the peak-matching algorithm. The peak-matching algorithm correctly identified a protein biomarker among as many as ∼1,400 possible bacterial proteins and gave rankings for protein identification comparable to the rankings obtained by more complicated and computationally intensive P value calculation. We often observed enhancement of the score for correct identification when results for MS-MS fragment ions were compared to results for residue-specific in silico fragment ions compared to non-residue-specific in silico fragment ions. In addition, the correctness of the algorithm''s identification was, in certain cases, further confirmed by fragment ion error analysis which compared random error caused by false matches between MS-MS fragment ions and in silico fragment ions with the systematic error observed for correct matches due to drift in the calibration of the TOF mass analyzer (38).(Portions of this work were presented at the 121st AOAC Conference [13] and at the 55th American Society of Mass Spectrometry Conference [14].)