Proteomic analysis of the photosystem I light-harvesting antenna in tomato (Lycopersicon esculentum)

Until now, more genes of the light-harvesting antenna of higher-plant photosystem I (PSI) than proteins have been described. To improve our understanding of the composition of light-harvesting complex I (LHCI) of tomato (Lycopersicon esculentum), we combined one- and two-dimensional (1-D and 2-D, respectively) gel electrophoresis with immunoblotting and tandem mass spectrometry (MS/MS). Separation of PSI with high-resolution 1-D gels allowed separation of five bands attributed to proteins of LHCI. Immunoblotting with monospecific antibodies and MS/MS analysis enabled the correct assignment of the four prominent bands to light-harvesting proteins Lhca1-4. The fifth band was recognized by only the Lhca1 antibody. Immunodetection as well as mass spectrometric analysis revealed that these protein bands contain not only the eponymous protein but also other Lhca proteins, indicating a heterogeneous protein composition of Lhca bands. Additionally, highly sensitive MS/MS allowed detection of a second Lhca4 isoform and of Lhca5. These proteins had not been described before on the protein level in higher plants. Two-dimensional gel electrophoresis revealed an even more diverse composition of individual Lhca proteins than was apparent from 1-D gels. For each of the four prominent Lhca proteins, four to five isoforms with different isoelectric points could be identified. In the case of Lhca1, Lhca4, and Lhca3, additional isoforms with slightly differing molecular masses were identified. Thus, we were able to detect four to ten isoforms of each individual Lhca protein in PSI. Reasons for the origin of Lhca heterogeneity are discussed. The observed variety of Lhca proteins and their isoforms is of particular interest in the context of the recently published crystal structure of photosystem I from pea, which showed the presence of only four Lhca proteins per photosystem I. These findings indicate that several populations of photosystem I that differ in their Lhca composition may exist.     

New method for peptide desorption from abundant blood proteins for plasma/serum peptidome analyses by mass spectrometry

This report describes a new method for desorption of low-molecular weight (LMW) peptides from abundant blood proteins for use in subsequent mass spectrometry analyses. Heating of diluted blood serum to 98°C for 15min resulted in dissociation of LMW peptides from the most abundant blood proteins. Application of blood plasma/serum fractionation using magnetic beads with a functionalized surface followed by heating of the resultant fractions significantly increases the number of LMW peptides detected by MALDI-TOF MS, enhances the general reproducibility of mass spectrometry profiles and considerably increases the number of identified blood serum peptides by LC-MS/MS using an Agilent 6520 Accurate-Mass Q-TOF.     

Mining the plasma proteome for disease applications across seven logs of protein abundance

The current state of proteomics technologies has sufficiently advanced to allow in-depth quantitative analysis of the plasma proteome and development of a related knowledge base. Here we review approaches that have been applied to increase depth of analysis by mass spectrometry given the substantial complexity of plasma and the vast dynamic range of protein abundance. Fractionation strategies resulting in reduced complexity of individual fractions followed by mass spectrometry analysis of digests from individual fractions has allowed well in excess of 1000 proteins to be identified and quantified with high confidence that span more than seven logs of protein abundance. Such depth of analysis has contributed to elucidation of plasma proteome variation in health and of protein changes associated with disease states.     

Identification and quantification of basic and acidic proteins using solution-based two-dimensional protein fractionation and label-free or 18O-labeling mass spectrometry

Reduction in sample complexity enables more thorough proteomic analysis using mass spectrometry (MS). A solution-based two-dimensional (2D) protein fractionation system, ProteomeLab PF 2D, has recently become available for sample fractionation and complexity reduction. PF 2D resolves proteins by isoelectric point (pI) and hydrophobicity in the first and second dimensions, respectively. It offers distinctive advantages over 2D gel electrophoresis with respects to automation of the fractionation processes and characterization of proteins having extreme pIs. Besides fractionation, PF 2D is equipped with built-in UV detectors intended for relative quantification of proteins in contrasting samples using its software tools. In this study, we utilized PF 2D for the identification of basic and acidic proteins in mammalian cells, which are generally under-characterized. In addition, mass spectrometric methods (label-free and 18O-labeling) were employed to complement protein quantification based on UV absorbance. Our studies indicate that the selection of chromatographic fractions could impact protein identification and that the UV-based quantification for contrasting complex proteomes is constrained by coelution or partial coelution of proteins. In contrast, the quantification post PF 2D chromatography based on label-free or 18O-labeling mass spectrometry provides an alternative platform for basic/acidic protein identification and quantification. With the use of HCT116 colon carcinoma cells, a total of 305 basic and 183 acidic proteins was identified. Quantitative proteomics revealed that 17 of these proteins were differentially expressed in HCT116 p53-/- cells.     

Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins

Serum analysis represents an extreme challenge due to the dynamic range of the proteins of interest, and the high structural complexity of the constituent proteins. In serum, the quantities of proteins and peptides of interest range from those considered "high abundance", present at 2-70% by mass of total protein, to those considered "low abundance", present at 10(-12) M or less. This range of analytical target molecules is outside the realm of available technologies for proteomic analysis. Therefore, in this study, we have developed a workflow toward addressing the complexity of these samples through the application of multidimensional separation techniques. The use of reversed-phase methods for the separation and fractionation of protein samples has been investigated, with the goal of developing an optimized serum separation for application to proteomic analysis. Samples of human serum were depleted of the six most abundant proteins, using an immunoaffinity LC method, then were separated under a variety of reversed-phase (RP) conditions using a macroporous silica C18 surface modified column material. To compare the qualities of the RP separations of this complex protein sample, absorbance chromatograms were compared, and fractions were collected for off-line SDS-PAGE and 2D-LC-MS/MS analysis. The column fractions were further investigated by determination of protein identities using either whole selected fractions, or gel bands excised from SDS-PAGE gels of the fractions. In either case samples underwent tryptic fragmentation and peptide analysis using MALDI-MS or LC-MS/MS. The preferred conditions for RP protein separation exhibited reproducibly high resolution and high protein recoveries (>98%, as determined by protein assay). Using the preferred conditions also permitted high column mass load, with up to 500 microg of protein well tolerated using a 4.6 mm ID x 50 mm column, or up to 1.5 mg on a 9.4 mm ID x 50 mm column. Elevated column temperature (80 degrees C) was observed to be a critical operational parameter, with poorer results observed at lower temperatures. The combination of sample simplification by immunoaffinity depletion combined with a robust and high recovery RP-HPLC fractionation yields samples permitting higher quality protein identifications by coupled LC-MS methods.     

Integrated mass spectrometry-based analysis of plasma glycoproteins and their glycan modifications

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.     

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.     

Analysis of the human serum proteome

Changes in serum proteins that signal histopathological states, such as cancer, are useful diagnostic and prognostic biomarkers. Unfortunately, the large dynamic concentration range of proteins in serum makes it a challenging proteome to effectively characterize. Typically, methods to deplete highly abundant proteins to decrease this dynamic protein concentration range are employed, yet such depletion results in removal of important low abundant proteins. A multi-dimensional peptide separation strategy utilizing conventional separation techniques combined with tandem mass spectrometry (MS/MS) was employed for a proteome analysis of human serum. Serum proteins were digested with trypsin and resolved into 20 fractions by ampholyte-free liquid phase isoelectric focusing. These 20 peptide fractions were further fractionated by strong cation-exchange chromatography, each of which was analyzed by microcapillary reversed-phase liquid chromatography coupled online with MS/MS analysis. This investigation resulted in the identification of 1444 unique proteins in serum. Proteins from all functional classes, cellular localization, and abundance levels were identified. This study illustrates that a majority of lower abundance proteins identified in serum are present as secreted or shed species by cells as a result of signalling, necrosis, apoptosis, and hemolysis. These findings show that the protein content of serum is quite reflective of the overall profile of the human organism and a conventional multidimensional fractionation strategy combined with MS/MS is entirely capable of characterizing a significant fraction of the serum proteome. We have constructed a publicly available human serum proteomic database (http://bpp.nci.nih.gov) to provide a reference resource to facilitate future investigations of the vast archive of pathophysiological content in serum. These authors contributed equally to this work.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

Protein Mobility Shifts Contribute to Gel Electrophoresis Liquid Chromatography Analysis

Profiling of cellular and subcellular proteomes by liquid chromatography with tandem mass spectrometry (MS) after fractionation by SDS-PAGE is referred to as GeLC (gel electrophoresis liquid chromatography)-MS. The GeLC approach decreases complexity within individual MS analyses by size fractionation with SDS-PAGE. SDS-PAGE is considered an excellent fractionation technique for intact proteins because of good resolution for proteins of all sizes, isoelectric points, and hydrophobicities. Additional information derived from the mobility of the intact proteins is available after an SDS-PAGE fractionation, but that information is usually not incorporated into the proteomic analysis. Any chemical or proteolytic modification of a protein that changes the mobility of that protein in the gel can be detected. The ability of SDS-PAGE to resolve proteins with chemical modifications has not been widely utilized within profiling experiments. In this work, we examined the ability of the GeLC-MS approach to help identify proteins that were modified after a small hairpin RNA-dependent knockdown in an experiment using stable isotope labeling by amino acids in cell culture-based quantitation.     

Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

Identification of p21Cip1 binding proteins by gel electrophoresis and capillary liquid chromatography microelectrospray tandem mass spectrometry

Proteins bound to a glutathione-S-transferase-p21Cip1 affinity column were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using tandem mass spectrometry. Capillary liquid chromatography coupled to microelectrospray tandem mass spectrometry (capLC-microESI MS/MS) in an ion trap allowed identification of the proteins present in the gel bands. Of eleven bands analyzed, fifty-three proteins were identified. More than one hundred tryptic peptides were detected on-line, automatically fragmented and used for protein characterization in databases. Samples were also analyzed by off-line nanospray and matrix-assisted laser desorption/ionization mass spectrometry. CapLC-microESI MS/MS was the most efficient technique for the analysis of these protein mixtures.     

Different immunoaffinity fractionation strategies to characterize the human plasma proteome

Plasma proteins may often serve as indicators of disease and are a rich source for biomarker discovery. However, the intrinsic large dynamic range of plasma proteins makes the analysis very challenging because a large number of low abundance proteins are often masked by a few high abundance proteins. The use of prefractionation methods, such as depletion of higher abundance proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may ultimately prove to be informative biomarkers. But there are few studies on comprehensive investigation of the proteins both in the fractions depleted and remainder. In the present study, two different immunoaffinity fractionation columns for the top-6 or the top-12 proteins in plasma were investigated and both the proteins in column-bound and flow-through fractions were subsequently analyzed. A two-dimensional peptide separation strategy, utilizing chromatographic separation techniques, combined with tandem mass spectrometry (MS/MS) was employed for proteomic analysis of the four fractions. Using the established HUPO PPP criteria, a total of 2401 unique plasma proteins were identified. The Multiple Affinity Removal System yielded 921 and 725 unique proteins from the flow-through and bound fractions, respectively, whereas the Seppro MIXED 12 column yielded identification of 897 and 730 unique proteins from the flow-through and bound fractions, respectively. When more stringent criteria, based on searching against the reversed database, were implemented, 529 unique proteins were identified from the four fractions with the confidence in peptide identification increased from 73.6% to 99%. To determine whether the presence of nontarget proteins in the immunoaffinity-bound fraction could be attributed to their interaction with high abundance proteins, co-immunoprecipitation analysis with an antibody to human plasma albumin was performed, which resulted in an identification of 40 unique proteins from the coimmunoprecipitate with the more stringent criteria. This study illustrated that combining the column-bound and flow-through fractions from immunoaffinity separation affords more extensive profiling of the protein content of human plasma. The presence of nontarget proteins in the column-bound fractions may be induced by their binding to the higher abundance proteins targeted by the immunoaffinity column.     

Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI MS/MS

A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.     

A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.     

Isoform analysis of LC-MS/MS data from multidimensional fractionation of the serum proteome

We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states.     

Characterization of protein variants and post-translational modifications: ESI-MSn analyses of intact proteins eluted from polyacrylamide gels

We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and post-translational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MSn analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MSn analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5-10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of kappa-casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of gel-eluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics.     

A survey of the plant mitochondrial proteome in relation to development

To expand the functional analysis of plant mitochondria, we have undertaken the building of the proteome of pea mitochondria purified from leaves (green and etiolated), roots and seeds. In the first stage, we focused our proteomic exploration on the soluble protein complement of the green leaf mitochondria. We used traditional two-dimensional polyacrylamide gel electrophoresis, in combination with size exclusion chromatography as a third dimension, to identify the major proteins and further resolve their macromolecular complexity. The two-dimensional map of soluble proteins of green leaf mitochondria revealed 433 spots (with Coomassie blue staining) and around 73% of the proteins (in mass) were identified using three different approaches: Edman degradation, matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry. Quite a lot of the polypeptides were present in multiforms which indicated the presence of isoforms or the occurrence of post-translational modifications. Among these proteins, we uncovered an abundant family that was identified as aldehyde dehydrogenases, representing approximately 7.5% of the soluble proteins. The comparative analysis of soluble mitochondrial proteomes led to the identification of a number of proteins which were specifically present in root or in seed mitochondria, thus revealing the impact of tissue differentiation at the mitochondrial level.     

An efficient extraction method to enhance analysis of low abundant proteins from soybean seed

Large amounts of the major storage proteins, β-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the low abundant proteins, different from the main storage protein fractions, in one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of isopropanol showed that greater than 30% isopropanol was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing isopropanol concentration above 40% resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.     

High resolution mass spectrometric alveolar proteomics: identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micropreparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products. The high resolution mass spectrometric proteome analysis should facilitate the unequivocal identification of subunits, aggregations, modifications and degradation products of surfactant proteins and hence contribute to the understanding of the mechanistic basis of lung disease pathogenesis.