Salmonella species continue to be commonly associated with cases of food-borne disease in developed countries. In the United States in 2001, the incidence per 100,000 people was highest for salmonellosis (15.1), followed by campylobacteriosis (13.8) and shigellosis (6.4). Enteric pathogens usually contaminate the surface of raw animal products during slaughter and primary processing (scalding, defeathering or dehiding, rinsing, cutting, mixing, and grinding, etc.) and can attach and/or reside in the regular and irregular surfaces of the skin, multiply and, thereafter, contaminate food preparation surfaces, hands and utensils. Trisodium phosphate (TSP) has been approved by the USDA as a sanitizer to reduce surface loads of Salmonella on chicken carcasses. A number of studies had demonstrated that TSP effectively removes surface contamination of carcasses by food-borne pathogens. However, very little scientific evidence is available which identifies the actual mechanisms of TSP antimicrobial activity and the response of food-borne pathogens exposed to TSP. This study examined both the physiological and molecular response of Salmonella enterica serovar Enteritidis to TSP treatment. The role of high pH during TSP treatment on its antimicrobial activity was examined. Adaptation of S. enterica serovar Enteritidis to TSP treatment was also examined by analyzing the proteome of serovar Enteritidis cells using two-dimensional gel electrophoresis and mass spectrometry. The role of high pH on the antimicrobial activity of TSP was examined using comparative studies involving treatment solutions containing different concentrations of TSP, treatment solutions adjusted to the equivalent pH as in each of the TSP treatments and TSP solutions pH adjusted to 7.0. Direct and indirect indices of cell survival, membrane damage, and cellular leakage were also employed to examine specific antimicrobial effects. Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH made with NaOH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or NaOH-alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. These results indicate that TSP is a potent membrane-acting agent, and provide compelling evidence that high pH during TSP treatment was responsible for its antimicrobial activity. Adaptation of S. enterica serovar Enteritidis with a sublethal concentration of TSP resulted in the induction of the alkaline stress response. Alkaline stress response involves induced thermotolerance, resistance to higher concentrations of TSP, high pH and sensitivity to acid. Examination of the proteome of TSP-adapted cells revealed differential expression of a number of proteins but did not include the common heat shock proteins involved in thermotolerance. However, TSP adaptation caused a shift in the membrane fatty acid composition from unsaturated to a higher saturated and cyclic fatty acid. This shift in fatty acid composition increases the melting point of the cytoplasmic membrane so that it remains functional at high temperatures. Biofilm bacteria are more resistant to sanitizers, heat and antimicrobial agents than their planktonic counterparts. Examination of the proteome of TSP-adapted biofilm cell of S. enterica serovar Enteritidis revealed little overlap in the protein profile compared to TSP-adapted planktonic cells. Proteomic examination of planktonic and biofilm cells of S. enterica serovar Enteritidis revealed differential expression of a number of proteins involved in DNA replication, stress survival and transport of newly synthesized proteins. These results clearly indicate that changes in the expression of specific genes are involved in the biofilm mode of growth, which could play a significant role in resistance to antimicrobial agents. The results of the current study provide a better understanding of the mechanisms of antimicrobial action of TSP and also elucidate the response of S. enterica serovar Enteritidis to TSP and high pH adaptation. The study also raises new questions regarding stress tolerance of S. Enteritidis following TSP or alkaline pH adaptation with relevance to food safety.

Salmonella species continue to be commonly associated with cases of food-borne disease in developed countries. In the United States in 2001, the incidence per 100,000 people was highest for salmonellosis (15.1), followed by campylobacteriosis (13.8) and shigellosis (6.4). Enteric pathogens usually contaminate the surface of raw animal products during slaughter and primary processing (scalding, defeathering or dehiding, rinsing, cutting, mixing, and grinding, etc.) and can attach and/or reside in the regular and irregular surfaces of the skin, multiply and, thereafter, contaminate food preparation surfaces, hands and utensils. Trisodium phosphate (TSP) has been approved by the USDA as a sanitizer to reduce surface loads of Salmonella on chicken carcasses. A number of studies had demonstrated that TSP effectively removes surface contamination of carcasses by food-borne pathogens. However, very little scientific evidence is available which identifies the actual mechanisms of TSP antimicrobial activity and the response of food-borne pathogens exposed to TSP. This study examined both the physiological and molecular response of Salmonella enterica serovar Enteritidis to TSP treatment. The role of high pH during TSP treatment on its antimicrobial activity was examined. Adaptation of S. enterica serovar Enteritidis to TSP treatment was also examined by analyzing the proteome of serovar Enteritidis cells using two-dimensional gel electrophoresis and mass spectrometry. The role of high pH on the antimicrobial activity of TSP was examined using comparative studies involving treatment solutions containing different concentrations of TSP, treatment solutions adjusted to the equivalent pH as in each of the TSP treatments and TSP solutions pH adjusted to 7.0. Direct and indirect indices of cell survival, membrane damage, and cellular leakage were also employed to examine specific antimicrobial effects. Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH made with NaOH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or NaOH-alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. These results indicate that TSP is a potent membrane-acting agent, and provide compelling evidence that high pH during TSP treatment was responsible for its antimicrobial activity. Adaptation of S. enterica serovar Enteritidis with a sublethal concentration of TSP resulted in the induction of the alkaline stress response. Alkaline stress response involves induced thermotolerance, resistance to higher concentrations of TSP, high pH and sensitivity to acid. Examination of the proteome of TSP-adapted cells revealed differential expression of a number of proteins but did not include the common heat shock proteins involved in thermotolerance. However, TSP adaptation caused a shift in the membrane fatty acid composition from unsaturated to a higher saturated and cyclic fatty acid. This shift in fatty acid composition increases the melting point of the cytoplasmic membrane so that it remains functional at high temperatures. Biofilm bacteria are more resistant to sanitizers, heat and antimicrobial agents than their planktonic counterparts. Examination of the proteome of TSP-adapted biofilm cell of S. enterica serovar Enteritidis revealed little overlap in the protein profile compared to TSP-adapted planktonic cells. Proteomic examination of planktonic and biofilm cells of S. enterica serovar Enteritidis revealed differential expression of a number of proteins involved in DNA replication, stress survival and transport of newly synthesized proteins. These results clearly indicate that changes in the expression of specific genes are involved in the biofilm mode of growth, which could play a significant role in resistance to antimicrobial agents. The results of the current study provide a better understanding of the mechanisms of antimicrobial action of TSP and also elucidate the response of S. enterica serovar Enteritidis to TSP and high pH adaptation. The study also raises new questions regarding stress tolerance of S. Enteritidis following TSP or alkaline pH adaptation with relevance to food safety.

Mass spectrometry is a versatile, powerful analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry is key component in expanding the capabilities of mass spectrometry beyond just a molecular weight detector. Another key component is the discovery of electrospray ionization allowing not only liquid samples to be ionized but also generation of multiply charged ions enabling mass spectrometry analysis of large biomolecules. The fragmentation pathway of ion during tandem mass spectrometry is highly dependent on the nature of the ion as well as the form of dissociation technique employed. To-date, no single form of ion or dissociation method can provide all the structure information needed; therefore, it is common to use multiple forms of ions, different charge carrier or modifications, with a variety of other dissociation techniques to generate complimentary information. Practically, it is not always easy to generate the desired form of ion via ionization methods and is one of the limitations. Gas-phase ion/ion reactions provide an easy approach in manipulation of ions, either through changing the ion type or covalent modifications, in the gas-phase with the goal of enhancing the capabilities of mass spectrometers for either molecular weight or structural analysis. In this dissertation, studies of new gas-phase ion/ion chemistry for biomolecules such as carbohydrates and phopho/sulfopeptides were performed, and exploration into mass spectrometry analysis of IgGs is discussed. <br><div> Ion/ion reactions with carbohydrates were investigated with the goal of finding a charge-transfer or covalent modification reaction which can increase the structural information of carbohydrates upon tandem mass spectrometry. No luck was achieved with charge transfer ion/ion reactions which increased the overall fragmentation information in tandem mass spectrometry. Novel gas-phase covalent chemistry was discovered where alkoxides were found to form ester and ethers. It was also discovered the aldehyde functional group at the reducing end of carbohydrates are susceptible to Schiff-base modifications. Schiff-base has been previously reported in peptides and this is the first time it has been discovered for carbohydrates.</div><div> In the next project a gas-phase approach for the rapid screening of polypeptide anions for phosphorylation or sulfonation based on binding strengths to guanidinium-containing reagent ions was developed. The approach relies on the generation of a complex via reaction of mixtures of deprotonated polypeptide anions with dicationic guanidinium-containing reagent ions and subsequent dipolar DC collisional activation of the complexes. The relative strengths of the electrostatic interactions of guanidinium with deprotonated acidic sites follows the order carboxylate</div><div> Hyaluronic acid, a linear carbohydrate polymer with repeating units of D-glucuronic acid and N-acetyl D-glucosamine, was found to exhibit unique properties in its electrospray ionization mass spectrum that was never seen before. Electrospray of hyaluronic acid in aqueous solution in the negative polarity presented an incredibly intriguing mass spectrum, which we termed “emerald city” consisting of max charge or max charge-1 anions of hyaluronic acid. This is the first biomolecule observed to have the capability to deprotonate at every acid site that is possible. These set of highly charge anions exhibits unique characteristics upon use as a charge inversion reagent to charged invert multiply protonated proteins. A max of thirty-three protons was transferred when myoglobin 24+ was charge-inverted to a max charge state of 9- in the negative mode. Further research should be conducted to fully understand this phenomenal and its possible utilities.<br></div><div> Lastly, mass spectrometry analysis of monoclonal antibodies was performed. Monoclonal antibodies are 150 kDa sized protein complexes and is a major area of interest for pharmaceutical industry. Mass spectrometry analysis of big proteins is an emerging area for mass spectrometry and is quite different compared to small and medium molecule analysis on the mass spectrometer. Detailed in the last chapter are methods developed for sample cleanup of immunoglobulin G as well as the application of q2 DDC for removal of loosely bound adducts to achieve sharper peaks in the mass spectrum. Studies of protein denaturation was also conducted with methods such as circular dichroism and differential ion mobility also employed. And finally, a photochemical reaction setup was shown to cleave twelve out of sixteen total disulfide bonds in the immunoglobulin G within seconds compared to traditional solution phase reactions which can take hours.<br></div>

<p> </p> <p>  Mass spectrometry (MS) plays an important role in nanomaterials research by facilitating the discovery of superatomic clusters and fullerenes, enabling the identification of atomically substituted clusters, and contributing to understanding mechanisms of cluster formation. In this dissertation, we used different mass spectrometry methods as well as ion soft-landing to address some of the ongoing challenges in cluster science. The first challenge is to extend the atom-by-atom substitution method, which is a promising strategy for designing new cluster-based materials, to a wider range of molecular clusters. Due to challenges associated with the synthesis, purification, and crystallization, this approach has been achieved only for a handful of gold and silver clusters. We extended this approach to enable the substitution of the first-row transitional metals into the core of Co6S8(PEt3)6 cluster, a well-defined metal chalcogenide superatomic cluster and a popular building block for designing novel 2D materials. This cluster is widely used in energy and electronic applications and is an excellent model system for computational studies of cluster-based materials. High-resolution MS analysis identified the formation of Co5MS8(PEt3)6+ (M=Mn, Fe, Ni) clusters, indicating that only Mn, Fe, and Ni atoms can be incorporated into the Co6S8 core using our synthetic method. A combination of mass spectrometric analysis and theoretical studies reaved that each heteroatom has different impact on the relative stability, core-ligand interaction, as well as optical, magnetic, and electrochemical properties of the cluster. </p> <p>Another challenge in the cluster science addressed in this work is the controlled activation of fully ligated clusters by ligand removal. Conventional activation methods such as thermolysis or chemical treatment do not provide sufficient control of the number of the removed ligands and often suffer from sintering of NCs as a result of excessive ligand removal and degradation of the destabilized NC cores. Using a specially-designed ion soft-landing instrument, we achieved a controlled removal of one or two phosphine ligands form the synthesized cobalt sulfide clusters using collision-induced dissociation (CID). The resulting fragments were mass selected and soft-landed onto surfaces. We found out that the reactivity of the fragment ions on surfaces may be controlled by altering the composition of the cluster core and ligand binding energy to the cluster. Although some of the fragments formed by removing one ligand including Co5FeS8(PEt3)5+ and Co6S8L(PPh3)5+ remain unreactive on surfaces, other fragments including Co6S8(PEt3)5+, Co5NiS8(PEt3)5+, and Co6S8(PEt3-xPhx) (x=1-2) undergo selective dimerization.  We observe that the reactivity of fragment ions produced by removing one surface ligand is controlled by the relative stability of the corresponding precursor ions towards fragmentation. In particular, fragment ions generated from more stable precursors undergo a selective dimerization reaction. In contrast, fragment ions produced from the least stable precursors remain largely unreactive on surfaces. In addition, we found that fragments generated by removing two surface ligands are highly reactive and undergo several nonselective reactions. This study demonstrates that fragment ions are unique building blocks that may undergo selective reactivity on surfaces to generate compounds that are difficult to prepare using conventional synthetic methods. We believe that the controlled preparation of fragment ions using ion soft-landing is a generalizable method to activate wide range of ligated nanoclusters which opens a direction for materials design and innovation.</p> <p>Finally, soft-landing of well-characterized redox-active polyatomic anions, PW12O403- (WPOM), was carried out to explore the distribution of pure mass selected anions on semiconducting vertically-aligned TiO2 nanotubes, which were used as a model system for 3D semiconductive materials. Energy dispersive X-ray (EDX) mapping analysis revealed that WPOMs form micron-size aggregates on top of the TiO2 NT and only penetrates 1-1.3 µm into the 10 µm-long nanotubes. This aggregation is attributed to the high resistance of TiO2. This is different from what we see on conductive substrates such as carbon nanotubes (CNTs) where a uniform distribution of ions is typically observed. This study provides valuable insight into the functionalization of porous semiconducting surfaces using mass selected ions for applications in energy storage and electronics.</p>

<div>The first chapter reports a study of aryl nitrene intermediates. Although extensively studied over the past 30 years, phenyl nitrenes have a propensity to undergo rearrangement reactions and form polymeric tars. This is in stark contrast to the phenyl carbenes which are known to undergo several important reactions to produce a library of useful organic compounds. One such reaction is the insertion of phenyl carbenes into a double bond to produce a cyclopropane moiety. If aryl nitrenes can be exploited to conjure a similar reactivity, they would be an excellent synthetic route to produce aziridine rings which are a crucial component of many natural products. This review chapter is a collection of all the efforts that have been made in this regard.</div><div><br></div><div>In the next chapter, the electronic effect of the azide functional group on an aromatic system has been investigated by using Hammett-Taft parameters obtained from the effect of azide-substitution on the gas-phase acidity of phenol. Gas-phase acidities of 3- and 4-azidophenol have been measured by using mass spectrometry and the kinetic method and found to be 340.8 ± 2.2 and 340.3 ± 2.0 kcal/mol respectively. The relative electronic effects of the azide substituent on an aromatic system have been measured by using Hammett-Taft parameters. The σF and σR values are determined to be 0.38 and 0.02 respectively, consistent with predictions based on electronic structure calculations. The values of σF and σR demonstrate that azide acts an inductively withdrawing group but has negligible resonance contribution on the phenol. In contrast, acidity values calculated for substituted benzoic acids gives values of σF = 0.69 and σR = -0.39, indicating that the azide is a strong  donor, comparable to that of a hydroxyl group. The difference is explained as being the result of “chimeric” electronic behavior of the azide, similar to that observed previously for the n-oxide moiety, which can be more or less resonance donating depending on the electronic effects of other groups in the system.</div><div><br></div><div>Phenyl nitrenes undergo bimolecular chemistry under very specific circumstances. For example, having an oxide substituent at the para position of the phenyl ring enables the formation of an indophenol product from a photocatalyzed reaction of the nitrene. Although, this reaction has been reported before, the mechanism involved in this reaction has not been fully understood. A two-electron mechanism involving electrophilic aromatic substitution reaction has been proposed in the literature, however we found evidence that did not support this theory. Instead, we find this reaction analogous to the popular Gibbs’ reaction whose single electron transfer mechanism has been extensively studied. The following chapter encompasses a study of the mechanism of the photolysis reaction to look for evidence of a single electron transfer similar to the Gibbs’ reaction.</div><div><br></div><div>As mentioned earlier, phenyl nitrenes have a proclivity to undergo rearrangement reactions instead of exhibiting bimolecular reactivity that can lead to useful products. One of the strategies to overcome this challenge is to spatially separate the two electrons of an open-shell singlet nitrene so as to minimize electron-electron repulsion. This separation can be achieved by delocalizing the individual electrons over multiple aromatic rings and heteroatoms which can act as radical stabilizers. In this chapter, a short review of literature that sets precedence for developing a unique heteroatom containing aromatic backbone to achieve the necessary stabilization is presented. Our efforts in synthesizing the model azide precursor compound have also been discussed.</div>

<p>High resolution tandem mass spectrometry (MSⁿ) coupled with various separation techniques, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), is widely used to analyze mixtures of unknown organic compounds. In a mass spectrometric analysis, analytes of interest are at first transferred into the gas phase, ionized (protonated or deprotonated) and introduced into the instrument. Tandem mass spectrometric experiments may then be used to gain insights into structure and reactivity of the analyte ions in the gas phase. The tandem mass spectral data are often compared to those reported in external databases. However, the tandem mass spectra obtained for protonated analytes may be markedly different from those in external databases because protonation site manifested during a mass spectrometric experiment can be affected by the ionization technique, ionization solvents and condition of the ion source. This thesis focuses on investigating the effects of instrumental conditions and analyte concentrations on the protonation sites of 4-aminobenzoic acid. Reactivities of radical species were also investigated. A modified bracketing method was developed and proton affinities of a series of mono- and biradicals of pyridine were measured. In another study, a <i>para</i>-benzyne analog was generated in both solution and the gas phase and its reactivities towards various neutral reagents in the gas phase were compared to those in solution.</p> <p> Chapter 2 discusses the fundamental aspects of the instruments used in this research. In chapter 3, the effects of residual moisture in linear quadrupole ion trap on the protonation sites of 4-aminobenzoic acid are considered. Chapter 4 focuses on the use of gas-phase ion-molecule reactions with trimethoxymethylsilane (TMMS) for the identification of the protonation sites of 4-aminobenzoic acid. Further, the effects of analyte concentration on the protonation sites of 4-aminobenzoic acid are considered. Chapter 5 introduces a modified bracketing method for the experimental determination of proton affinities of a series of pyridine-based mono- and biradicals. In chapter 6, successful generation of <i>para</i>-benzynes in solution is discussed. The reactivity of a <i>para</i>-benzyne analog, 1,4-didehydrophenazine, is compared to its reactivity in the gas phase.</p>

<div>Mass spectrometry (MS) is a powerful and versatile analytical tool, especially for identification and analysis of complex mixtures. Coupling to high-performance liquid chromatography (HPLC) or gas chromatography (GC) provides additional dimension for mixture analysis. MS manipulates ionized analytes and separates them based on their mass-to-charge (<i>m/z</i>) ratios. MS is capable of providing molecular weight (MW) information by generating pseudo-molecular ions of the analytes. Detailed elemental compositions can be also obtained if high resolution MS is used. MS can also provide extensive structural information of the analyte ions. One of the most commonly used technique is tandem mass spectrometry (MSⁿ). Ions of interest are isolated and subject to sequential reactions (reactions with other molecules or dissociation reactions) to generate product ions that can provide structural information. MS is also a powerful tool for generating and studying highly reactive reaction intermediates, such as organic polyradicals.</div><div><br></div><div>The research described in this dissertation mainly focuses on the generation and gas-phase reactivity studies of different organic biradicals. Their reactions with various organic reagents are studied, and the reactivity-controlling factors are discussed. For example, the reactivity of several substituted pyridine-based biradical cations with 2,6-topology are discussed (all with singlet ground states), and their special reactivity from their excited triplet states are illustrated. Besides, several quinoline-based biradicals and cyano-substituted pyridine-based <i>para</i>-benzyne cations are also discussed. Some of the radicals (or ions) described in this dissertation are generated for the first time, i.e. the quinoline-based oxenium cations. Their structural characterization and gas-phase reactivity toward some organic molecules are discussed in the dissertation. Further, an electrophilic anion, <i>closo</i>-[B₁₂X₁₁]^- (X = Cl, Br) and its application in the activation of C-H and C-C bonds in hydrocarbon molecules are described in the dissertation.</div>

The function of many biological processes depends on the structure and composition of the biomolecules involved. Both spectroscopy and mass spectrometry provide complimentary information regarding the three-dimensional conformation and the composition, respectively, as well as many other things. Here, double resonance conformer specific spectroscopy coupled with the latest ab inito computational methods is used to make structural assignments at the atomic resolution as well obtain information regarding propensities of intramolecular interactions. Additionally, rapid cooling in conjunction with IR excitation to modulate and measure the relative populations of conformers present in the expansion. Two different designer peptide systems are studied, including an achiral acylated 𝛼-aminoisobutryic acid dipeptide (Ac-AIB2-R) with various C-terminal protecting groups (R=NHBn, NHBnF, 𝛼-methylbenzylamine) and an acylated 𝛾4-phenylalanine (Ac-𝛾4Phe-NHMe) with the a methyl amine C-terminal protecting group. Mass spectrometry is used to determine the kinetics of gas-phase covalent tagging reactions used to enhance the sequence coverage. The covalent modification reactions utilize click chemistry between NHS or HOBt substituted sulfobenzoic acid tags with nucleophiles present on the residues of the amino acids composing the backbone. Effective temperatures are approximated using the Tolmachev model, which relates the statistical average internal energy of the molecule to a temperature.

<div> <div> <div> <p>Understanding protein-protein interactions (PPIs) in a cell is essential for learning protein functions, pathways, and mechanisms of diseases. This dissertation introduces the computational method to predict PPIs. In the first chapter, the history of identifying protein interactions and some experimental methods are introduced. Because interacting proteins share similar functions, protein function similarity can be used as a feature to predict PPIs. NaviGO server is developed for biologists and bioinformaticians to visualize the gene ontology relationship and quantify their similarity scores. Furthermore, the computational features used to predict PPIs are summarized. This will help researchers from the computational field to understand the rationale of extracting biological features and also benefit the researcher with a biology background to understand the computational work. After understanding various computational features, the computational prediction method to identify large-scale PPIs was developed and applied to Arabidopsis, maize, and soybean in a whole-genomic scale. Novel predicted PPIs were provided and were grouped based on prediction confidence level, which can be used as a testable hypothesis to guide biologists’ experiments. Since affinity chromatography combined with mass spectrometry technique introduces high false PPIs, the computational method was combined with mass spectrometry data to aid the identification of high confident PPIs in large-scale. Lastly, some remaining challenges of the computational PPI prediction methods and future works are discussed. </p> </div> </div> </div>

The sensitivity, speed, and reproducibility of modern mass spectrometers enable in-depth new functional looks into the cellular proteome. Thousands of proteins can be detected in a single sample. In Co-Fractionation Mass Spectrometry (CF-MS) method, the input sample is fractionated by any biochemical method of choice. The reduced complexity of each fractionated sample leads to better proteome coverage. The separation profiles provide functional information on the proteins. This application has been used to predict organelle localization based on co-purification with marker proteins. More recently, CF-MS is being used to measure the apparent masses and determine the localization of soluble or membrane-associated protein complexes. This Ph.D. dissertation focuses on the extension of the boundary of CF-MS application to learn how protein complex evolution and protein complex composition have been accomplished. In the first part of this dissertation, the data will be presented on the degree to which variation in protein oligomerization across plant species is present, how proteomics in phylogenetic analysis (phyloproteomics/evolutionary proteomics) helps understand the evolutionary changes, and how oligomerization drives neofunctionalization during plant evolution. The latter part will describe that CF-MS coupled with multiple orthogonal chromatographic separations increases the resolving power of the profiling technique, enabling the composition of protein complexes to be predicted in the subaleurone layers of rice endosperm. Lots of novel protein complexes involved in RNA binding protein, translation, and the tissue-species metabolism will be discussed.