WO2005103069A1 - Multidimensional protein separation - Google Patents

Abstract

In large scale proteome applications, protein separation is paramount to observing discrete changes and quantitative evaluation must coincide with qualitative protein identification for effective differential analysis. A four dimensional (4D) platform for resolving and differentially analyzing complex biological samples is presented. The system, collectively termed CAX-PAGE/RPLC-MSMS, combines bi-phasic ion-exchange chromatography (1st dimension) and polyacrylamide gel electrophoresis (2nd dimension) for protein separation, quantification and differential band targeting leading toward subsequent capillary reverse phase liquid chromatography (3rd dimension) and data dependant tandem mass spectrometry (4th dimension) for semi-quantitative and qualitative peptide analysis.

Description

MULTIDIMENSIONAL PROTEIN SEPARATION

FIELD OF THE INVENTION [0001] The invention relates to the field of proteomics. In particular, a system and methods for identification and quantification .of proteins and peptides from complex biological samples is provided.

BACKGROUND OF INVENTION [0002] From the recent completion of human and other species genomes it has become apparent that many biological systems operate through changes at the protein level not • governed by gene regulation (Denslow N et al, J. Neurotrauma (2003) 20, 401-407). The new field of proteomics has arisen to provide a more complete picture of cell operation at the protein level under normal and challenged conditions. The pervasive influence of proteomic technology has been rapid, however many challenges still persist. Of primary concern is the inherent complexity of biological protein mixtures: the shear number of proteins (10,000 or more) and the wide dynamic range of concentration for example. To handle this challenge, most research laboratories have relied on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation (Griffin T. j. et al. J. Bio. Chem. (2001) 276, 45497-45500. Peng J. et al. J. Mass Spectrom. (2001) 36, 1083-1091). There are numerous limitations to this technology that have prompted researchers to look elsewhere in particular difficulties with gel-to-gel reproducibility, dynamic range, pi range, the ability to resolve very small and large proteins and those that are hydrophobic in nature restrict the use of2D-PAGE.

[0003] The use of ion-exchange has been limited to that of a pre-fractionation step for separating a particular group or proteins, i.e., a clean-up method for analysis of a subset of proteins from mixtures. Ion-exchange has also been incorporated prior to reverse phase separations for peptide analysis post enzymatic digestion. This typically incorporates an acidic modifier to shift the charge distribution to allow more peptides to adhere to the ion- exchange support. In any case, a large number of proteins or peptides from a complex mixture are not retained by ion-exchange columns as they are either opposite to or neutral in charge relative to proper operating conditions.

[0004] An urgent need thus exists in the art for the to separate, identify and purify complex biological mixtures. SUMMARY [0005] Differential proteomic analysis has arisen as a large scale means to discern proteome wide changes upon treatment, injury or disease. In large scale proteome applications, protein separation is paramount to observing discrete changes. In addition, quantitative evaluation must coincide with qualitative protein identification for effective differential analysis. A four dimensional (4D) platform for resolving and differentially analyzing complex biological samples is presented.

[0006] In a preferred embodiment, separation and differential analysis of proteins and/or peptides in a crude biological sample comprises a method based on four independent physical properties and two complimentary quantification methods are employed. The platform, collectively termed CAX-PAGE/RPLC-MSMS, combines bi-phasic ion-exchange chromatography (1^st dimension) and polyacrylamide gel electrophoresis (2^nd dimension) for protein separation, quantification and differential band targeting leading toward subsequent capillary reverse phase liquid chromatography (3^r dimension) and data dependant tandem mass spectrometry (4 dimension) for semi-quantitative and qualitative peptide analysis. [0007] In another preferred embodiment, a method of isolating and quantifying biomarkers, comprises obtaining a crude biological sample; subjecting the sample to a bi- phasic ion-exchange chromatography and obtaining fractions; separating the fractions by polyacrylamide gel electrophoresis into bands according to molecular weight; cutting bands from the polyacrylamide gel; subjecting the separated bands to capillary reverse phase liquid chromatography and obtaining a second set of fractions; and, subjecting the second set of fractions to tandem mass spectrometry; thereby, isolating and quantifying the isolated biomarkers.

[0008] In another preferred embodiment, a method of isolating, quantifying biomarkers comprises obtaining a crude biological sample(s); clarifying the sample(s) via centrifugation and ultrafiltration; subjecting the samples sequentially to bi-phasic ion-exchange chromatography and obtaining fractions; separating fractions by polyacrylamide gel electrophoresis into bands according to molecular weight and quantitatively imaging band density and evaluating protein expression; cutting selected bands from the polyacrylamide gel and subjecting them to in-gel digestion; subjecting the digested bands to capillary reverse phase liquid chromatography in tandem with mass spectrometry; thereby, isolating, quantifying and identifying the biomarker associated peptides.

[0009] In another preferred embodiment, the ion-exchange chromatography comprises at least a plurality of gradients, preferably, the ion exchange chromatography comprises at least a two step gradient, preferably, the ion exchange chromatography comprises a three step gradient, preferably, the ion exchange chromatography comprises a five step gradient, preferably, ion exchange chromatography comprises a ten step gradient, preferably, the ion exchange chromatography comprises between about a two step gradient up to a twenty step gradient.

[00010] In another preferred embodiment, the ion-exchange chromatography comprises a plurality of ion exchange media. Preferably, the media comprises weak anionic and/or cationic exchange media and strong anionic and/or cationic media.

[00011] In another preferred embodiment, the bi-phasic ion ion-exchange chromatography comprises at least a two step gradient, preferably the bi-phasic ion exchange chromatography comprises a three step gradient. Two step gradient comprise linear transitions from 0% to about 15% in a volume of about 12 mL. Three step gradients comprise a linear transition from about 15% to about 50% in a volume of about 7 mL, held at about 50% in a volume of about 2 mL and re-equilibrated to 0% in about 1 mL volume.

[00012] In another preferred embodiment, the two-step gradient comprises a linear transition from 0% to about 15% in a volume of about 12 mL up to 50 mL.

[00013] In another preferred embodiment, the three-step gradient comprises a linear transition from about 15% to about 50% in a volume of about 7 mL up to 50 mL, held at about 50% in a volume of about 2 mL up to 50 mL and re-equilibrated to 0% in about 1 mL up to 50 mL volume.

[00014] In accordance with the invention, the gradient is optimized depending on the viscosity ofthe mixture, the complexity ofthe biological sample and the like and can include a plurality of gradients.

[00015] In another preferred embodiment, the polyacrylamide gel comprises a gradient of between about 1% up to 50% and/or can be a gel without a gradient. The percentage ofthe gel can be from about 1% to about 50%.

[00016] In accordance with the invention, the bands on the gel can be visualized using any number of dyes. For example, Coomassie blue, silver staining, Sypro Ruby, cyanine dyes and the like.

[00017] In a prefeπed embodiment, the bands are subjected to enzymatic digestion in-gel.

Alternatively, the bands are excised and subjected to enzymatic digestion. The preferred enzymes include, but not limited to hydrolases - these include esterases, carbohydrases, nucleases, deaminases, amidases, and proteases; Hydrases such as fumarase, enolase, aconitase and carbonic anhydrase; oxidases, dehydrogenases; transglycosidases; transphosphorylases and phosphomutases; transaminases; transmethylases; transacetylases; desmolases; isomerases; ligases. Preferably, the enzyme is a tryptase. [00018] In another prefeπed embodiment, the enzyme digested bands are subjected to reverse phase liquid chromatography. Preferably, the ri_c values ofthe reverse phase liquid chromatography are between about 100 to about 250.

[00019] In another prefeπed embodiment, the fractions eluted from the reverse phase liquid chromatography are further subjected to tandem mass spectrometry and separated by mass-to-charge. Preferably, the r-_c values are at least about 1 x 10⁵ up to 1 x 10¹⁰. [00020] h another prefeπed embodiment, a method of isolating and quantifying proteins and/or peptides comprises obtaining a crude biological sample(s); clarifying the sample(s) via centrifugation and ultrafiltration; subjecting the samples sequentially to bi-phasic ion- exchange chromatography and obtaining fractions; separating fractions by polyacrylamide gel electrophoresis into bands according to molecular weight and quantitatively imaging band density and evaluating protein expression; cutting selected bands from the polyacrylamide gel and subjecting them to in-gel digestion; subjecting the digested bands to capillary reverse phase liquid chromatography in tandem with mass spectrometry; thereby, isolating, quantifying and identifying the peptides.

[00021] In another prefeπed embodiment, the ion-exchange chromatography comprises at least a plurality of gradients, preferably, the ion exchange chromatography comprises at least a two step gradient, preferably, the ion exchange chromatography comprises a three step gradient, preferably, the ion exchange chromatography comprises a five step gradient, preferably, ion exchange chromatography comprises a ten step gradient, preferably, the ion exchange chromatography comprises between about a two step gradient up to a twenty step gradient.

[00022] In another prefeπed embodiment, the ion-exchange chromatography comprises a plurality of ion exchange media. Preferably, the media comprises weak anionic and/or cationic exchange media and strong anionic and/or cationic media, for example Waters Protein Pak, Pharmacia's Source Q, etc.

[00023] hi another prefeπed embodiment, the bi-phasic ion ion-exchange chromatography comprises at least a two step gradient, preferably the bi-phasic ion exchange chromatography comprises a three step gradient. Two step gradient comprise linear transitions from 0% to about 15% in a volume of about 12 mL. Three step gradients comprise a linear transition from about 15% to about 50% in a volume of about 7 mL, held at about 50% in a volume of about 2 mL and re-equilibrated to 0% in about 1 mL volume. [00024] In another prefeπed embodiment, the two-step gradient comprises a linear transition from 0% to about 15% in a volume of about 12 mL up to 50 mL.

[00025] In another prefeπed embodiment, the three-step gradient comprises a linear transition from about 15% to about 50% in a volume of about 7 mL up to 50 mL, held at about 50% in a volume of about 2 mL up to 50 mL and re-equilibrated to 0% in about 1 mL up to 50 mL volume.

[00026] In another prefeπed embodiment, the bi-phasic ion ion-exchange chromatography comprises at least a plurality of gradients, preferably, the bi-phasic ion exchange chromatography comprises at least a two step gradient, preferably, the bi-phasic ion exchange chromatography comprises a three step gradient, preferably, the bi-phasic ion exchange chromatography comprises a five step gradient, preferably, bi-phasic ion exchange chromatography comprises a ten step gradient, preferably, the bi-phasic ion exchange chromatography comprises between about a two step gradient up to a twenty step gradient.

[00027] In accordance with the invention, the gradient is optimized depending on the viscosity ofthe mixture, the complexity ofthe biological sample and the like and can include a plurality of gradients.

[00028] In another prefeπed embodiment, the polyacrylamide gel comprises a gradient of between about 1% up to 50% and/or can be a gel without a gradient. The percentage ofthe gel can be from about 1% to about 50%.

[00029] In accordance with the invention, the bands on the gel can be visualized using any number of dyes. For example, Coomassie blue, silver staining, Sypro Ruby, cyanine dyes and the like.

[00030] In a prefeπed embodiment, the bands are subjected to enzymatic digestion in-gel.

Alternatively, the bands are excised and subjected to enzymatic digestion. The prefeπed enzymes include, but not limited to hydrolases - these include esterases, carbohydrases, nucleases, deaminases, amidases, and proteases; Hydrases such as fumarase, enolase, aconitase and carbonic anhydrase; oxidases, dehydrogenases; transglycosidases; transphosphorylases and phosphomutases; transaminases; transmethylases; transacetylases; desmolases; isomerases; ligases. Preferably, the enzyme is a tryptase.

[00031] In another prefeπed embodiment, the enzyme digested bands are subjected to reverse phase liquid chromatography. Preferably, the ri_c values ofthe reverse phase liquid chromatography are between about 100 to about 250. [00032] In another prefeπed embodiment, the fractions eluted from the reverse phase liquid chromatography are further subjected to tandem mass spectrometry and separated by mass-to-charge. Preferably, the n_c values are at least about 1 x 10⁵upto 1 x 10¹⁰. [00033] Accordingly, in one embodiment, the subject invention pertains to a method of identifying at least one biomarker comprising obtaining a biological sample from a patient known to have an injury, disorder or pathological condition (test sample(s)); obtaining at least one biological sample from a patient known not to have such injury or pathological condition (control sample(s)); sequentially performing CAX chromatography to said biological samples to produce fraction samples; subjecting fraction samples to electrophoresis in a gel; visualizing proteins in said gel; identifying presence of proteins in one sample not present in another sample, wherein differential presence indicates a biomarker candidate. Preferably, subjecting fraction samples to electrophoresis comprises performing 1-D PAGE. Also prefeπed is running electrophoresis with fractions from the test sample side-by-side with coπesponding fractions from the control sample. Visualizing the proteins may comprise staining fractions from the control sample with a first dye and staining fractions from the test sample with a different dye. The coπesponding fraction samples may be overlaid whereby different colors generated indicate the presence of a protein in one or the other sample, or both. The method of identifying biomarkers can be applied to identify biomarkers relating to, but not limited to neurological injuries, disorders and diseases; cancer; autoimmune disorders; stress; exposure to toxins; and joint disease. In the case of identifying biomarkers for brain injury, blood, serum or central spinal fluid samples from individuals known to have a brain injury are compared to individuals known not to have a brain injury. Generally speaking, the sample may be tissue homogenate, urine, blood, CSF, serum or other biological fluid present in the body.

[00034] In a prefeπed embodiment, a method of isolating and differential quantitative analysis of proteins and/or peptides in complex biological mixtures, said method comprising: obtaining a crude biological sample; subjecting the sample to a bi-phasic ion-exchange chromatography and obtaining fractions; running the fractions obtained in order of elution side-by-side on a polyacrylamide gel electrophoresis allowing for differential comparison; quantifying bands obtained by polyacrylamide gel electrophoresis by densitometric scanning; selecting bands which are differentially expressed at least about two-fold as compared to a normal control; digesting the differentially expressed bands with enzyme; subjecting the enzyme digested bands to capillary reverse phase liquid chromatography online in tandem with mass spectrometry; thereby, isolating and quantifying the isolated proteins and/or peptides. Preferably, quantification of isolated proteins is validated by comparing the protein amounts with gel band density. [00035] In another prefeπed embodiment, the bi-phasic ion ion-exchange chromatography comprises at least a plurality of gradients, preferably, the bi-phasic ion exchange chromatography comprises at least a two step gradient, preferably, the bi-phasic ion exchange chromatography comprises a three step gradient, preferably, the bi-phasic ion exchange chromatography comprises a five step gradient, preferably, bi-phasic ion exchange chromatography comprises a ten step gradient, preferably, the bi-phasic ion exchange chromatography comprises between about a two step gradient up to a twenty step gradient. [00036] ' In accordance with the invention, the gradient is optimized depending on the viscosity ofthe mixture, the complexity ofthe biological sample and the like and can include a plurality of gradients.

[00037] In another prefeπed embodiment, the polyacrylamide gel comprises a gradient of between about 1% up to 50% and/or can be a gel without a gradient. The percentage ofthe gel can be from about 1% to about 50%.

[00038] In accordance with the invention, the bands on the gel can be visualized using any number of dyes. For example, Coomassie blue, silver staining, Sypro Ruby, cyanine dyes and the like.

[00039] In another prefeπed embodiment, bands on the gel digested by enzymes selected from the group consisting of hydrolases, esterases, carbohydrases, nucleases, deaminases, amidases, proteases, hydrases, fumarase, enolase, aconitase carbonic anhydrase, oxidases, dehydrogenases; transglycosidases; transphosphorylases phosphomutases, transaminases; transmethylases, transacetylases, desmolases, isomerases; and ligases. Preferably, the enzyme is a tryptase.

[00040] In another prefeπed embodiment, the enzyme digested bands are subjected to reverse phase liquid chromatography. Preferably, the n_c values ofthe reverse phase liquid chromatography are between about 100 to about 250.

[00041] In another prefeπed embodiment, the fractions eluted from the reverse phase liquid chromatography directly flow into the mass spectrometry and separated by mass-to- charge. Preferably, the r-_c values are at least about 1 x 10⁵, preferably, the n_c values are about 1 x 10⁶, preferably, the n_c values are about 1 x 10⁷, preferably, the n_c values are about 1 x 10^s, preferably, the r-_c values are about 1 x 10⁹, preferably, the ri_c values are about 1 x 10¹⁰. [00042] Other aspects ofthe invention are described infra. BRIEF DESCRIPTION OF DRAWINGS [00043] The invention is pointed out with particularity in the appended claims. The above and further advantages of this invention may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which: [00044] Figure 1 shows ion-exchange chromatograms of 1 mg of rat cerebellum brain tissue lysate separated with a NaCI gradient: strong-cationic ion-exchange (SCX) separation of tissue lysate; strong-anionic ion-exchange (SAX) separation of tissue lysate; tandem catiomc and anionic ion-exchange (CAX) separation of tissue lysate. Timing ofthe two stage gradient is as indicated.

[00045] Figure 2 shows a rat cerebellum proteome visualized on ID-PAGE following CAX fractionation. MKR indicates molecular weight markers.

[00046] Figure 3 A shows a chromatogram of rat cerebellum brain lysate (1 mg protein) run sequentially in triplicate by CAX.

[00047] Figure 3B is a gel showing selected fractions (paired as indicated) from three replicate CAX runs resolved and visualized side-by-side on ID-PAGE. Protein compliment remained constant while band intensity varied on average by only 6%. . [00048] Figure 4 shows a chromatogram of rat cerebellum tissue lysate (750 μg) performed with SCX, SAX, and CAX with two step elution processes. [00049] Figures 5A-5B shows a comparison of rat cerebellum and cortex proteomes via sequential CAX and side-by-side ID-PAGE. Figure 5A is a chromatogram showing an overlay of cerebellum and cortex CAX chromatograms at 280 nm. Figure 5B shows a side- by-side pairing of 25 fractions run on ID-PAGE. (M = cerebellum; X = cortex). Boxed bands were excised for protein identification. Note letter labeling for coπelation with Tables 1 and 2.

[00050] Figure 6 shows a colorized rat cerebellum-cortex differential proteome display after C AX-PAGE. The colorized display was performed by overlaying adjacent lanes from Figure 5B.

[00051] Figures 7A and 7B show 2D-DIGE differential display of rat cerebellum-cortex. Figure 7A is a false color overlay of cortex Cy3 (green) and cerebellum Cy5 (red) labeled DIGE images. Figure 7B shows the results of 2D differential software analysis comparing cortex and cerebellum tissue. Spots with 100% difference between samples are indicated by yellow for greater in cortex and green for greater in cerebellum, while blue indicates spots found only in one sample. DETAILED DESCRIPTION [00052] A system and methods for resolution, identification and quantitation of complex biological mixtures are provided. In particular, the system comprises combined cationic and anionic exchange in tandem with gel electrophoresis to enable the rapid and efficient identification of proteins and/or peptides such as biomarkers indicative of a disease state. Furthermore, the invention provides protein visualization techniques that enable rapid identification of differential expression, or presence of, certain proteins in a biological sample relating to a certain biological or medical condition.

Definitions

[00053] Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter. [00054] The term "capillary" as used in reference to the electrophoretic device in which electrophoresis is carried out in the methods ofthe invention is used for the sake of convenience. The term should not be construed to limit the particular shape ofthe cavity or device in which electrophoresis is conducted. In particular, the cavity need not be cylindrical in shape. The term "capillary" as used herein with regard to any electrophoretic method includes other shapes wherein the internal dimensions between at least one set of opposing faces are approximately 2 to 1000 microns, and more typically 25 to 250 microns. An example of a non-tubular aπangement that can be used in certain methods ofthe invention is the a Hele-Shaw flow cell. Further, the capillary need not be linear; in some instances, the capillary is wound into a spiral configuration, for example.

[00055] As used herein, the term "ion exchange efficiency" means the efficiency with which ions in a solution are exchanged with those bound to an ion exchange material. For example, ion exchange efficiency can be defined as E/M, where E is the percent of ions in a solution that are exchanged with the ions bound to an ion exchange resin, and M is the mass ofthe ion exchange resin. Ion exchange efficiency can be determined by, for example, passing equal volumes of water containing equal ion concentrations through the ion exchange media being measured, and then measuring how many ofthe ions have been exchanged. Ion exchange can easily be determined by methods known to those skilled in the art including, but not limited to, ultraviolet and visible absorption measurements, atomic absoφtion spectra, and titration. Therefore, the plurality of ion-exchange media used in the invention are easily determined based on desired ion exchange efficiencies. Ion exchange media are available through commercial sources. [00056] "Marker" or "biomarker" in the context ofthe present invention refers to a polypeptide (of a particular apparent molecular weight) which is differentially present in a sample taken from patients having a disease, such as cancer, injury such as neural injury and/or neuronal disorders as compared to a comparable sample taken from control subjects (e.g., a person with a negative diagnosis, normal or healthy subject). [00057] The phrase "differentially present" refers to differences in the quantity and/or the frequency of a protein and/or peptides present in a sample taken from patients having for example, neural injury as compared to a control subject. For example, a marker can be a polypeptide which is present at an elevated level or at a decreased level in samples of patients with neural injury compared to samples of control subjects. Alternatively, a marker can be a polypeptide which is detected at a higher frequency or at a lower frequency in samples of patients compared to samples of control subjects. A marker can be differentially present in terms of quantity, frequency or both.

[00058] A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is statistically significantly different from the amount ofthe polypeptide in the other sample. For example, a polypeptide is differentially present between the two samples if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, or if it is detectable in one sample and not detectable in the other. [00059] Alternatively or additionally, a polypeptide is differentially present between the two sets of samples if the frequency of detecting the polypeptide in samples of patients' is statistically significantly higher or lower than in the control samples. For example, a polypeptide is differentially present between the two sets of samples if it is detected at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% more frequently or less frequently observed in one set of samples than the other set of samples.

[00060] "Diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The ^v "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[00061] A "crude biological sample" as used herein is any sample, for example, tissue, cell etc which is not subjected to any type of treatment but refers to for example, a homogenized tissue sample, a lysed cell and the like.

[00062] Anion exchangers can be classified as either weak or strong. As used herein, a weak anion exchange medium" or "weak cationic exchanger" is one where the charge group is a weak base, which becomes deprotonated and, therefore, loses its charge at high pH. DEAE-cellulose is an example of a weak anion exchanger, where the amino group can be positively charged below pH ~ 9 and gradually loses its charge at higher pH values. A "strong anion exchanger" on the other hand, contains a strong base, which remains positively charged throughout the pH range normally used for ion exchange chromatography (pH 1-14). [00063] Cation exchangers can also be classified as either weak or strong. A "strong cation exchange medium" or "strong cation exchanger"contains a strong acid (such as a sulfopropyl group) that remains charged from pH 1 - 14; whereas a "weak cation exchange medium" or "weak cationic exchanger" contains a weak acid (such as a carboxymethyl group), which gradually loses its charge as the pH decreases below 4 or 5. [00064] The charge on the protein affects its behavior in ion exchange chromatography. Proteins contain many ionizable groups on the side chains of their amino acids as well as their amino - and carboxyl - termini. These include basic groups on the side chains of lysine, arginine and histidine and acidic groups on the side chains or glutamate, aspartate, cysteine and tyrosine. The pH ofthe solution, the pK ofthe side chain and the side chain's environment influence the charge on each side chain. The relationship between pH, pK and charge for individual amino acids can be described by the Henderson-Hasselbalch equation: φH -pK* log [conjugate base]; [eonju ateaeidl i

[00065] In general terms, as the pH of a solution increases, deprotonation ofthe acidic and basic groups on proteins occur, so that carboxyl groups are converted to carboxylate anions (R-COOH to R-COO-) and ammonium groups are converted to amino groups (R- NH³⁺ to R-NH₂). In proteins the isoelectric point (pi) is defined as the pH at which a protein has no net charge. When the pH > pi, a protein has a net negative charge and when the pH < pi, a protein has a net positive charge. The pi varies for different proteins. Isolation and Quantitation

[00066] The methods ofthe present invention utilize a combination of methods conducted in series to resolve mixtures of proteins. The methods are said to be conducted in series because the sample(s) isolated in each method are from solutions or fractions containing proteins isolated in the preceding method, with the exception ofthe sample electrophoresed in the initial method. As used herein, the terms protein, peptide and polypeptide are used interchangeably and refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues of coπesponding naturally-occurring amino acids, including amino acids which are modified by post-translational processes (e.g., glycosylation and phosphorylation). [00067] In a prefeπed embodiment, the present invention relates to a system and methodology for identifying protein patterns associated with predetermined biological characteristics. Another aspect relates to a system and methodology for identifying protein patterns associated with predetermined clinical parameters. A further aspect relates to a system and methodology for identifying protein patterns associated with predetermined medical conditions. Still, a further aspect relates to a system and methodology for identifying protein patterns associated with predetermined diseases.

[00068] In another prefeπed embodiment, the present invention also relates to a system and methodology for predicting the existence or non-existence of at least one predetermined biological characteristic. The present invention also relates to a system and methodology for predicting the presence of disease in an animal body, such as a mammal. [00069] In other prefeπed embodiments, a system and methodology for rapidly identifying proteins associated with disease or other biological conditions are used as biomarkers in diagnostic applications. The present invention also relates to a system and methodology for using biomarker proteins as a therapeutic target for treatment of disease or other biological conditions. The present invention also relates to a system and methodology for discovering proteins that are useful as imaging or therapeutic targets of disease. [00070] In another prefeπed embodiment, protein biomarkers are identified for monitoring the course of a disease, and for determining appropriate therapeutic intervention. Additional features ofthe invention will be set forth in part in the description which follows, and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice ofthe invention.

[00071] As a non-limiting example intended for illustration pvnposes, enhanced tandem cationic/anionic ion-exchange chromatography increased protein retention to 88% for uniform protein distribution across 25 or more fractions per sample. Paired fractions from each sample were loaded on conventional ID-PAGE for differential comparison with a loading reproducibility of 94% while avoiding gel-to-gel variability issues. The CAX-PAGE theoretical peak capacity of 3570, extendable to 7600, was on par with other 2D protein separations; however resolving power is further extended using subsequence peptide separations. From a differential target list based on a two fold band intensity difference between samples, matched bands were in-gel digested and separated by capillary reverse phase liquid chromatography inline with a quadruple ion trap tandem mass spectrometer.

The 4D theoretical peak capacity is about 1.43x10 , with a saturation factor of only 3.5% assuming a peptidome of 5x10⁵ proteins fragmented into 100 unique peptides. Differential analysis by CAX-PAGE/RPLC-MSMS is effectively demonstrated using a neuroproteomic model comparing cerebellum and cortex rat tissues. Protein separations revealed 137 distinct differential targets, 67% more than with alternative 2D-DIGE technology, of which 33 were randomly selected for subsequent peptide analysis. Verifiable protein identification was determined in 85% of cases, out of which 89% had semi-quantitative peptide data validating differential CAX-PAGE band intensity determinations. Further, matching gel band and identified protein masses from 16 to 273 kDa coπoborated protein determination and demonstrated the platform's effective mass range.

[00072] According to one embodiment, the invention combines cationic and anionic exchange separation, herein termed "CAX chromatography", with the resolving and visualization power of ID-PAGE. Complex protein mixtures, nearly all biological samples, can be resolved by ionic-strength then mass by this multi-dimensional separation technique. Low salt and surfactant concentrations can be tolerated, while samples with high salt or surfactants can be pre-cleaned by dialysis or precipitation procedures. The sample is then injected onto tandem cationic and anionic columns or a mixed bed column (containing both media). The low percentage of neutral proteins run through the column(s) and are collected in the first few fractions. The retained proteins are eluted by increasing the counter-ion salt concentration in a gradient fashion with fraction collection. For example, the gradient is a matter of proportioning two or more mobile phases together. As an illustrative example, 20mM Tris Buffered water and B: 1M salt (NaCI) in 20mM Tris Buffered water are mixed proportionally from 0% to 100% B in multiple linear gradients - performed through computer control of two pumps pushing at different rates (one for each mobile phase). The strategy with CAX optimization is that gradient segments can be added (e.g.,1-20 segments) each with a different rate of mixing (i.e., gradient slope) to allow even separation of proteins across the fractions collected. Different sample types (e.g. tissue vs. biofluids) requires such optimization. Samples can also be collected at different flow rates (0.010 mL/min to 2.5 mL/min) based mainly on column size (2 mm to 2 cm i.d.). For example, as few as 9 to as many as 50 fractions per sample can be collected, with volumes from 100 μl to 2 ml. [00073] In accordance with the invention, a gradient step is as small as 2.5% change in A:B to as much as 50% change in as little as 1 min to as long as 60 min. Therefore, gradient steps can be tailored to the sample at hand.

[00074] Any type of ion-exchange material can be used. For example, ion exchange resins can be cationic, anionic, mixtures of cation and anionic, or biologically related. Examples of ion exchange resins useful in this invention include, but are not limited to, those made of cross-linked polyvinylpyrolodone and polystyrene, and those having ion exchange functional groups such as, but not limited to, halogen ions, sulfonic acid, carboxylic acid, iminodiacetic acid, and tertiary and quaternary amines. Specific examples of cationic ion exchange resins include, but are not limited to: AMBERJET™ 1200(H); Amberiite™ CG-50, IR-120(plus), IR-120(plus) sodium form, IRC-50, IRC-50S, and IRC-718; Amberlyst™ 15, 15 (wet), 36(wet), A-21, A-26 borohydride, bromide, chromic acid, fluoride, and tribromide; and DOWEX™ 50WX2-100, 50WX2-200, 50WX2-400, 50WX4-50, 50WX4-100, 50WX4- 200, 50WX4-200R, 50WX4-400, HCR-W2, 50WX8-100, 50WX8-200, 50WX8-400, 650C, MARATHON™ C, DR-2030, HCR-S, MSC-1, 88, CCR-3, MR-3, MR-3C, and Retardion™. Specific examples of anionic ion exchange resins include, but are not limited to: AMBERJET™ 4200(CI); Amberiite™ IRA-67, IRA-400, IRA-400(CI), IRA-410, IRA-743, IRA-900, LRP-64, IRP-69, XAD-4, XAD-7, and XAD-16; AMBERSORB™ 348F, 563, 572 and 575; DOWEX™ 1X2-100, 1X2-200, 1X2-400, 1X4-50, 1X4-100, 1X4-200, 1X4-400, 1X8-50, 1X8-100, 1X8-200, 1X8-400, 21K CI, 2X8-100, 2X8-200, 2X8-400, 22 CI, MARATHON™ A, MARATHON™ A2, MSA-1, MSA-2, 550A, 66, MARATHON™ WBA, and MARATHON™ WGR-2; and Merrifield's peptide resins. A specific example of mixed cationic and anionic resins is Amberiite™ MB-3A. Specific examples of biologically related resins that can be used in the processes and products ofthe invention include, but are not limited to, Sephadex™ CM C-25, CM C-50, DEAE A-25, DEAE A-50, QAE A-25, QAE A-50, SP C-25, and SP C-50. These cationic, anionic, mixed cationic and anionic, and biologically related ion exchange resins are commercially available from, for example, Aldrich Chemical Co., Milwaukee, Wis., or from Rohm and Haas, Riverside, N.J. Additional examples of ion exchange resins include, but are not limited to AG-50W-X12, Bio-Rex™ 70, and Chelex™ 100, all of which are tradenames of Bio-Rad, Hercules, Calif. [00075] Examples of functional groups used in ion exchange chromatography for selection of weak vs. strong anionic or cationic media are as follows: Functional Group pK Value Characteristic Description TMAE-Group pK > 13 strongly basic Trimethylammoniumethyl- DEAE-Group pK 11 weakly basic Diethylaminoethyl- DMAE-Group pK 8-9 weakly basic Dimethylaminoethyl- COO-Group pK4.5 weakly acidic Carboxy- S03-Group pK < 1 strongly acidic Sulfoisobutyl- SE-Group pK < 1 strongly acidic Sulfoethyl-

[00076] A variety of buffers at different pH values (e.g., Tris-HCL, HEPES, and multi-pH phosphate buffers) can be used to tailor charge distribution. Additionally, a pH gradient can be used in place ofthe salt gradient mentioned here - this would be more akin to isoelectric focusing used in 2D-PAGE. The benefit of a salt gradient is that all proteins can be maintained at the same pH, preferably neutral, to prevent denaturing. Fractions are then concentrated down with micro-spin tubes, to which gel electrophoresis sample buffer is added for reconstitution and collected for direct loading onto one-dimensional polyacrylamide gel electrophoresis (ID-PAGE). The gels are then visualized with traditional protocols. A variety of conventional staining techniques, such as but not limited to, Coomassie stain for detection of high-concentration proteins, or more sensitive stains (e.g., silver or Sypro ruby) for detection of less abundant proteins, may be used in accord with the principles ofthe invention. This method is refeπed to herein as CAX/1D-PAGE. [00077] In another prefeπed embodiment, the CAX / ID-PAGE system is used for differential comparison of complex biological mixtures. Two strategies were performed to demonstrate differential proteomic analysis. The first utilized the reproducibility of CAX / ID-PAGE to run two different samples sequentially (e.g., a control and a treated sample) by CAX chromatography and then load paired fractions side-by-side on ID-PAGE. Though differences can be observed by close examination of adjacent lanes, a visualization method was developed to observe expression differences by a positive (green) or negative (red) color shift from equal expression (yellow) to take advantage ofthe human eye's keen ability to detect color. A second differential expression strategy utilizes cyanine dye technology in a similar fashion to that applied with 2D-PAGE. This embodiment has the advantage of being more reproducible as both control and treated samples can be mixed and run through CAX / ID-PAGE separation together since they can be visualized via different fluorescence conditions. However, cyanine dyes are less sensitive and more difficult to use than other stains such as silver or sypro ruby for visualization of less concentrated proteins. Both strategies are useful depending on the experiment. Those skilled in the art will appreciate that various dyes may be implemented in accordance with the teachings herein. [00078] In accordance with the invention, linear gels can be from about 4% acrylamide to about 18% acrylamide (e.g. 4,5,6,7.5,8,10,12,12.5,14,15,16,18%). Gradient gels can be in differing gradients such as for example: 4-15%, 4-20%, 8-16%, 10.5-14%, 10-20%. Any size gel can be used, for example commercially available gels are about 20cm SDS-PAGE with differing numbers of gel lanes (10 to 26 wells) and gel thicknesses (1mm to 1.5mm). [00079] In another embodiment, CAX is implemented in combination with second dimensional liquid chromatography for separation of proteins and peptides. As discussed supra, ion-exchange chromatography has been used as a first stage to multi-dimensional chromatography. In both cases, sample fractionation can be enhanced by employing CAX chromatography in place of either cationic (SCX) or anionic (SAX) ion-exchange chromatography alone. This is based on the same principle as illustrated in the Examples which follow, that all acidic and basic molecules will bind with CAX. This embodiment shows superior, unexpected results when conducting online 2D-LC separations for performing shotgun proteomics or for analysis of post-translationally modified (PTM) proteins, particularly for those proteins/peptides that are modified with highly charged groups (e.g., phosphate). Such PTMs can be further elucidated with special stains (that are selective to PTMs of interest. The present invention permits high-resolution separation of complex protein mixtures, particularly biological samples derived from tissue, body fluids, and all forms of cell lysates, with visualization using conventional gel stains. [00080] In another prefeπed embodiment, differential proteome analysis of complex protein mixtures - the comparison of protein expression between two samples (e.g., biomarker discovery, sub-proteome analysis, etc.) is conducted using the methods ofthe invention. This technology is also used to visualize post-translationally modified subproteomes by use of special selective gel stains.

[00081] In another prefeπed embodiment, the CAX chromatography is placed online with additional liquid chromatography (e.g., reverse phase, size exclusion, etc.) to provide increased sample fractionation and two dimensional resolution. This can be applied to both protein and peptide mixtures, including those with post-translationally modified components with separation at the preparatory down to the capillary scale. The CAX chromatography is used to pre-fractionate a complex mixture for multiple ID-PAGE and/or 2D-PAGE analysis. [00082] Accordingly, in one embodiment, the subject invention pertains to a method of identifying at least one biomarker comprising obtaining a biological sample from a patient known to have an injury, disorder or pathological condition (test sample(s)); obtaining at least one biological sample from a patient known not to have such injury or pathological condition (control sample(s)); sequentially performing CAX chromatography to said biological samples to produce fraction samples; subjecting fraction samples to electrophoresis in a gel; visualizing proteins in said gel; identifying presence of proteins in one sample not present in another sample, wherein differential presence indicates a biomarker candidate. Preferably, subjecting fraction samples to electrophoresis comprises performing 1-D PAGE. Also prefeπed is running electrophoresis with fractions from the test sample side-by-side with coπesponding fractions from the control sample. Visualizing the proteins comprises staining fractions from the control sample with a first dye and staining fractions from the test sample with a different dye. The coπesponding fraction samples may be overlaid whereby different colors generated indicate the presence of a protein in one or the other sample, or both. (See, the Examples which follow). The method of identifying biomarkers can be applied to identify biomarkers relating to, but not limited to neurological injuries, disorders and diseases; cancer; autoimmune disorders; stress; exposure to toxins; and joint disease. In the case of identifying biomarkers for brain injury, blood, serum or central spinal fluid samples from individuals known to have a brain injury are compared to individuals known not to have a brain injury. The sample may be tissue homogenate, urine, blood, CSF, serum or other biological fluid present in the body.

[00083] ' The markers identified by the methods taught herein may be used to diagnosed multiple medical conditions, including but not limited to, brain injuries, such as those caused by accidental trauma, strokes, etc.; presence of tumors; autoimmune diseases; and neurodegenerative diseases. Furthermore, the CAX chromatography methods may be used as research techniques for basic research. U.S. Patent applications 20040066955; 20030232396; and 20030211531; and PCT publication WO 2002/US0019813 discuss methods of identifying biomarkers.

[00084] ^λ In another prefeπed embodiment, a method of isolating and differential quantitative analysis of proteins and/or peptides in complex biological mixtures, said method comprising: obtaining a crude biological sample; subjecting the sample to a bi-phasic ion- exchange chromatography and obtaining fractions; running the fractions obtained in order of elution side-by-side on a polyacrylamide gel electrophoresis allowing for differential comparison; quantifying bands obtained by polyacrylamide gel electrophoresis by densitometric scanning; selecting bands which are differentially expressed at least about twofold as compared to a normal control; digesting the differentially expressed bands with enzyme; subjecting the enzyme digested bands to capillary reverse phase liquid chromatography online in tandem with mass spectrometry; thereby, isolating and quantifying the isolated proteins and/or peptides. Preferably, quantification of isolated proteins is validated by comparing the protein amounts with gel band density.

[00085] Accordingly, the bi-phasic ion ion-exchange chromatography comprises at least a plurality of gradients, preferably, the bi-phasic ion exchange chromatography comprises at least a two step gradient, preferably, the bi-phasic ion exchange chromatography comprises a three step gradient, preferably, the bi-phasic ion exchange chromatography comprises a five step gradient, preferably, bi-phasic ion exchange chromatography comprises a ten step gradient, preferably, the bi-phasic ion exchange chromatography comprises between about a two step gradient up to a twenty step gradient. In accordance with the invention, the gradient is optimized depending on the viscosity ofthe mixture, the complexity ofthe biological sample and the like and can include a plurality of gradients.

[00086] In another prefeπed embodiment, the polyacrylamide gel comprises a gradient of between about 1% up to 50% and/or can be a gel without a gradient. The percentage ofthe gel can be from about 1% to about 50% and the gradient can be changed to isolate bands of close molecular weights during the differential analysis.

[00087] In accordance with the invention, the bands on the gel can be visualized using any number of dyes. For example, Coomassie blue, silver staining, Sypro Ruby, cyanine dyes and the like.

[00088] In another preferred embodiment, bands on the gel digested by enzymes selected from the group consisting of hydrolases, esterases, carbohydrases, nucleases, deaminases, amidases, proteases, hydrases, fumarase, enolase, aconitase carbonic anhydrase, oxidases, dehydrogenases; transglycosidases; transphosphorylases phosphomutases, transaminases; transmethylases, transacetylases, desmolases, isomerases; and ligases. Preferably, the enzyme is a tryptase.

[00089] In another prefeπed embodiment, the enzyme digested bands are subjected to reverse phase liquid chromatography. Preferably, the ri_c values ofthe reverse phase liquid chromatography are between about 100 to about 250.

[00090] In another prefeπed embodiment, the fractions eluted from the reverse phase liquid chromatography directly flow into the mass spectrometry and separated by mass-to- charge. Preferably, the ri_c values are at least about 1 x 10⁵, preferably, the ri_c values are about 1 x 10⁶, preferably, the n_c values are about 1 x 10⁷, preferably, the rt_c values are about 1 x 10⁸, preferably, the r-_c values are about 1 x 10⁹, preferably, the r-_c values are about 1 x 10¹⁰. [00091] An illustrative example, without limiting the invention in any way, of differential analysis is as follows: The potential of CAX-PAGE is realized with its ability to provide differential expression maps for subsequent targeted differential RPLC-MSMS analysis. As a test case, proteomic differences between cerebellum and cortex regions of rat brain were explored. It was expected that the compliment of proteins would be similar in both tissues, but that expression would differ. Clear chromatographic differences in Figure 5 a are observed between cerebellum and cortex lysates sequentially separated by CAX. For differential analysis, fractions from each run are paired and run side-by-side on ID-PAGE (Figure 5b), whereby problems of gel-to-gel reproducibility are avoided by always comparing matching fractions on the same gel. Side-by-side fraction pairing as in Figure 5b allows for direct visualization of differential expression using simple, cost efficient, visible stains (e.g., Coomassie Blue, silver, Deep Purple). Fluorescent stains such as Sypro Ruby also work well, though they require a more expensive fluorescence scanner (three times the cost for the liquid chromatography station). Whether visible or fluorescent stains are used, images are easily assessed with the Phoretix ID software. Automatic processing is performed to identify gel lanes, providing a clear boundary along the x-axis. Band height is also distinguishable, though fainter bands tend to require manual verification. Band intensity is automatically calculated along with band mass based on calibration with a traditional protein marker. Data is then output to an excel spreadsheet with adjacent bands lined up between lanes. A threshold set at 100% difference in band intensity is applied to generate a list of target bands for further analysis, thereby minimizing mass spectrometry workload in comparison with shotgun proteomic protocols.

Samples

[00092] The methods of the invention can be used with a wide range of sample types. Essentially any protein-containing sample can be utilized with the methods described herein. The samples can contain a relatively small number of proteins or can contain a large number of proteins, such as all the proteins expressed within a cell or tissue sample, for example. [00093] In prefeπed embodiments, tissue and cell culture samples are clarified of large cellular debris - clumps of cell parts that are not easily broken up. This can be done by any method such as described in the Examples which follow. For example, centrifugation and running the supernatant through a 0.1 μm centrifugal filter. Generally, nothing else need be done (no need to remove salts, or mix in other compounds), though highly viscous liquids may require dilution prior to loading e.g. tissue lysate, cell culture lysate, and CSF. [00094] Samples can be obtained from any organism or can be mixtures of synthetically prepared proteins or combinations thereof. Thus, suitable samples, can be obtained, for example, from microorganisms (e.g., viruses, bacteria and fungi), animals (e.g., cows, pigs, horses, sheep, dogs and cats), hominoids (e.g., humans, chimpanzees, and monkeys) and plants. The term "subject" as used to define the source of a sample includes all ofthe foregoing sources, for example. The term "patient" refers to both human and veterinary subjects. The samples can come from tissues or tissue homogenates or fluids of an organism and cells or cell cultures. Thus, for example, samples can be obtained from whole blood, serum, semen, saliva, tears, urine, fecal material, sweat, buccal, skin, spinal fluid, tissue biopsy or necropsy and hair. Samples can also be derived from ex vivo cell cultures, including the growth medium, recombinant cells and cell components. In comparative studies to identify potential drug or drug targets, one sample can be obtained from diseased cells and another sample from non-diseased cells, for example.

Variations of Analysis

[00095] The methods ofthe invention use any variety of analyses for quantitation. For example, densitometric analysis, infra-red spectroscopy, nuclear magnetic resonance spectroscopy, UV/VIS spectroscopy and complete or partial sequencing. Coupling the electrophoresis to any mass spectroscopy (MS) methods is within the scope ofthe invention. A variety of mass spectral techniques can be utilized including several MS/MS methods and Electrospray-Time of Flight MS methods. Such methods can be used to determine at least a partial sequence for proteins resolved by the methods such as a protein sequence tag.

Advantages

[00096] As mentioned above, CAX chromatography can be used in conjunction with 20- PAGE analysis. CAX chromatography/1-D PAGE has certain advantages over the use of 2D-PAGE alone. For example, the CAX gradient can be optimized to provide even fractionation of proteins, or to emphasize a particular area. The number of fractions and associated gel lanes is limited only by the amount of time needed to process the samples — this means that resolution can be expanded indefinitely to provide significantly higher separation of proteins over the limited format size of 2D-PAGE. Many ID-PAGE acrylamide compositions can be used to emphasize resolution at higher, lower, or intermediate protein mass. 2D-PAGE is limited in format. All acidic and basic proteins are separated by CAX prior to second dimensional separation, as compared to the limited pi range of isoelectric focusing strips used for 2D-PAGE. The increased resolving power of CAX/1D-PAGE provides easier visualization of low and high concentration proteins — greater dynamic range based on increased resolving capability. Hydrophobic proteins are retained and separated by CAX / ID-PAGE. Components such as Urea or Chaps do not need to be added to perform CAX. Reproducibility is improved as only side-by-side lanes need be compared by ID-PAGE and distortion is primarily in one direction. The use of a salt gradient over a pH gradient allows proteins to be kept in their native state. [00097] The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more ofthe features ofthe previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations ofthe invention will become apparent to those skilled in the art upon review ofthe specification. The scope ofthe invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. [00098] All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention.

EXAMPLES Materials and Methods Sample Preparation.

[00099] Male Sprague Dawley rats (five) purchased from Harlan (Indianapolis, IN) were acclimated for 7 days prior to sacrificing. The rats were then anesthetized with 4% isoflurane in a carrier gas of 1 : 1 O₂/N₂O (4 minutes), and were perfused with 0.9% saline transcardially prior to decapitation via guillotine. Cerebellum and cortex brain regions were dissected and transfeπed to microfuge tubes kept on dry ice. Sections were snap frozen in liquid nitrogen then ground to a fine powder via mortar and pestle kept on dry ice. Powder was scrapped into chilled microfuge tubes to which 0.1% SDS lysis buffer (300 μl) was added containing 150 mM NaCI, 3 mM EDTA, 2 mM EGTA, 1% IGEPAL (all from Sigma- Aldrich, St. Louis, MO), one tablet of Complete Mini Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) and 1 mM sodium vanadate (Fisher Scientific, Fair Lawn, NJ) with the sample solution brought to neutral pH using Tris-base (Sigma- Aldrich). Cell lysis was conducted over 3 hours at 4 °C with hourly vortexing. Lysates were spun down at 14,000 φm at 4 °C for 10 minutes to remove DNA, lipids, and particulates. Supernatants were then filtered through 0.1 μm Millipore Ultrafree-MC filters (Bedford, MA) for further clarification. Protein concentrations were determined via Bio-Rad DC Protein Assay (Hercules, CA), after which pooled (n=5) 1 mg cortex and 1 mg cerebellum samples were prepared for differential comparison.

Ion-Exchange Chromatography.

[000100] A Bio-Rad (Hercules, CA) Biologic DuoFlow system with QuadTec UV detector and BioFrac fraction collector was used with Uno series SAX (Ql) and SCX (SI) pre-packed ion exchange columns. For CAX chromatography, SI and Ql columns were placed in series. Buffers consisted of ice cold 20 mM Tris-HCl (pH 7.5 molecular biology grade, Fisher Scientific) in HPLC water (Burdick & Jackson, Muskegon, MI)(mobile phase A). A two step elution gradient was performed with 1 M NaCI (Fisher Scientific, crystalline 99.8% certified) (mobile phase B) at a flow rate of 1 mL/min with a linear transition from 0 to l5% B in l2.5 mL followed by 15 to 50% B in 7 mL. The composition was then held at 50% B for 2 mL, and then re-equilibrated to 0% B in 1 mL. An optimized three step gradient was used for differential analysis. At a flow rate of 1 mL/min, the first linear transition was from 0 to 5% B in 2.5 mL then from 5% to 15% in 9 mL followed by 15% to 50% in 10 mL. Again the composition was held at 50% B for 2 mL, and re-equilibrated to 0% B in 1 mL. UN chromatograms were collected at a wavelength of 280 nm. Twenty-five 1 mL fractions were autonomously collected via the BioFrac fraction collector into 1.5 mL screw cap microfuge tubes (RPI, Mt. Prospect, IL) kept on ice.

1D-SDS-PAGE.

[000101] Fractions collected during ion exchange chromatography were concentrated via Millipore YM-30 centrifugal filters. Each fraction was spun through filters pre- washed with 500 μl of HPLC water as two 500 μl sequential portions at 13,500 rpm for 20 minutes. Laemmli sample buffer (Bio-Rad, with 5% BME) was added to the retentate, and incubated for 10 minutes prior to collection by centrifugation at 3,500 rpm for 3 minutes. The supernatant for each fraction was boiled at 85 °C for two minutes, and then loaded onto Invitrogen Νovex 10 - 20% gradient 1 mm 10 well gels in a Tris-glycine buffer system (Carlsbad, CA) alongside a lane of Amersham Biosciences Rainbow Marker (Piscataway, ΝJ) for initial studies. Differential analysis between cerebellum and cortex tissues was performed by pairing fractions for loading side-by-side (i.e., cerebellum fraction 1 next to cortex fraction 1, etc.) on Bio-Rad Criterion 10 - 20% gradient 1 mm 18 well gels in a Tris- glycine buffer system.

Protein Retention and Recovery of CAX. [000102] Protein recovery was evaluated with SAX, SCX, and combined phase CAX. A constant protein amount (750 μg) in a 100 μl injection ofthe previously described cerebellum lysate was loaded. A 1 mL/min isocratic flow was maintained for 9 minutes to allow unretained proteins to flow through. The mobile phase composition was then increased in 1 minute and held for 9 minutes at 50% B to elute bound proteins collected in a normal gradient run. This was followed by an additional increase to 100% B in 1 minute which was held for another 9 minute to check for additional protein. Throughout, UN absorbance was monitored at 280 nm and 1 mL fractions were collected, each concentrated using Millipore YM-30 centrifugal filters and analyzed via Bio-Rad DC Protein Assay.

CAX-PAGE Coomassie Blue Imaging.

[000103] Gels were visualized by regressive staining using concentrated Bio-Rad Coomassie Blue R250 for 20 minutes and destained in 40% HPLC grade ethanol (EM Science, Gibbstown, ΝJ) / 10% acetic acid (ACS Plus grade, Fisher) for approximately two hours. Images were captured with an Epson 1640 XL flatbed scanner (Long Beach, CA) and saved as 8-bit TIFF files. Differential analysis of Coomassie blue stained gelswas performed using Phoretix ID (Nonlinear Dynamics, Newcastle, UK) gel image analysis software. Band intensities were automatically calculated and manually verified for bands above a preset threshold. Intensities were output to Excel (Microsoft, Redmond, WA) for differential evaluation. Manual confirmation was aided by superimposing cerebellum lanes false colored red over adjacent cortex lanes false colored green creating gradient color lanes for each fraction. Image contrast was improved by adjusting RGB color balance to emphasize mid tones over shadows.

2D-DIGE.

[000104] Cerebellum and cortex samples (1 mg each) were prepared from the same pooled material used for CAX-PAGE. Each was adjusted to 2% SDS, followed by TCA precipitation. The pellet was air dried and resuspended in 150 μl of pH 8.8 urea lysis buffer. Benzonase Nuclease (Novagen, Madison, WI) and 5 mM magnesium chloride (Fisher) were added, incubating the mixture for 30 minutes on ice to degrade nucleic acids. The solution was clarified by centrifugation with a Beckman Coulter (Fullerton, CA) Airfuge at 100,000 g for 30 minutes. The supernatant was dialyzed against the urea lysis buffer overnight at room temperature. A 50 μg portion of cortex and cerebellum lysate was labeled with Cy3 and Cy5 minimal dyes (Amersham Biosciences), respectively, using the manufacturer's suggested protocol. Cyanine labeled samples were combined with 275 μg both of unlabeled cortex and cerebellum lysates. The solution was adjusted to 0.2% of IPG pH 3 to 10 buffer (Amersham Biosciences) and 100 mM DTT with a trace of Orange G stain (Fisher). An 18 cm non-linear pH 3 to 10 IPG strip (Amersham Biosciences) was rehydrated in the mixed sample under oil overnight at room temperature. Proteins were focused on the strip at 8 kV until migration was complete (65 kVhrs). Proteins in the strip were reduced with 100 mM DTT in the reaction buffer 50 mM pH 6.8 Tris-HCl, 6 M Urea, 30% glycerol, and 2% SDS. Alkylation was performed with 2.5% iodoacetamide in the same reaction buffer. The strip was mounted atop a Bio-Rad precise 8-16% Tris glycine gel, and run for 6 hrs at 25 mA and 24 °C. Separate Cy3 and Cy5 images were collected on an Amersham Typhoon 8600 fluorescence imager, and processed with Phoretix 2D software (Nonlinear Dynamics).

In-Gel Digestion.

[000105] Gels were thoroughly rinsed with HPLC water. Target differential bands were excised and dissected into four cubes and placed in 0.5 μl tubes. Each was washed with HPLC water, then 50% 100 mM ammonium bicarbonate (Fisher) / 50% acetonitrile (Burdick- Jackson, HPLC grade). Pieces were dehydrated with 100% acetonitrile and dried by speedvac (ISS110, Thermo Savant, Milford, MA). Cubes were rehydrated with 50 μl of 10 mM dithiothreitol (Calbiochem, San Diego, CA) in 50 mM ammonium bicarbonate and incubated for 30 minutes at 56 °C. Dithiothreitol was replaced by 50 μl of 55 mM iodoacetamide (Calbiochem) in 50 mM ammonium bicarbonate, and reacted for 30 minutes in the dark at room temperature. Gel pieces were washed with 50 mM ammonium bicarbonate, and dehydrated with 100% acetonitrile followed by speedvac. Rehydration was performed with 15 μl of a 12.5 ng/μl trypsin solution (Promega Gold, Madison, WI) for 30 minutes at 4 °C, then 20 μl of 50 mM ammonium bicarbonate was added and left at 37 °C overnight for digestion. The supernatant along with two 50% acetonitrile/5% acetic acid extractions were placed into a new tube. The peptide extract was dried by speedvac and resuspended in 20 μl 4% acetonitrile/0.4% acetic acid. Capillary RPLC-MSMS.

[000106] Capillary RPLC tandem ion trap mass spectrometry was employed for protein identification as described previously with some modifications. Nanoflow reverse phase chromatography was performed with a 100 μm i.d. x 5 cm capillary column packed in-house with Agilent (Palo Alto, CA) 3 μm C-18 particles behind an Upchurch 0.5 μm PEEK microfilter assembly. The integrated polymerized frit was replaced with a pulled emitter made from 25 μm i.d. capillary affixed to the other end ofthe microfilter assembly. Thirty minute gradients, 4% HPLC acetonitrile/0.4% acetic acid (Fisher, Optima grade) to 60% acetonitrile/0.4% acetic acid, were used to elute tryptic peptides. Tandem mass spectra were collected on a ThermoElectron (San Jose, CA) LCQ Deca XP-Plus using data-dependant analysis. Collected data were searched against the trypsin indexed complete NCBI RefSeq mammalian database filtered for rat taxonomy using ThermoElectron Bioworks Browser (version 3.1). We report protein identifications made with two or more peptides matched with strict cross coπelation values of Xc >1.8, 2.5, and 3.5 for +1, +2, and +3 charge states, respectively. Data filtering was performed with DTASelect, and cerebellum vs. cortex MSMS data was compared using Contrast software.

Example 1: CAX Chromatography — First Dimension.

[000107] The majority of proteins in biological samples such as tissue lysates or body fluids retain regions of significant charge on their external surfaces when at physiological pH. Considered together, the net charge of these external regions approximately half of the time is negative and somewhat less than half of the time is net positive. Though in reality regions of external charge act independent of net charge, a general explanation for performing combined SCX and SAX is that categorically CAX will retain positively and negatively charged proteins rather than predominantly those of one net polarity. Figure 1 illustrates the difference in gradient separation of a complex brain tissue lysate with independent SCX, SAX, and CAX chromatography. The single ion exchangers allow a significant portion of the proteome to flow through unretained, as evidenced by the large peak at the begimiing of the chromatograms. CAX binds most proteins by charge interaction leaving generally net neutral proteins that despite regions of low charge density will not bind in the presence of background counter ions. In practice, a portion of this flow through fraction is resolved through hydrophobic interaction into approximately 4 flow-through fractions. Figure 1 shows tandem catiomc anionic ion-exchange (CAX) chromatography with superior protein retention. Strong-cationic ion-exchange (SCX), strong-anionic ion-exchange (SAX), and tandem cationic and anionic ion-exchange (CAX) chromatograms are shown overlaid for 1 mg of rat cerebellum brain lysate. Chromatograms are identically scaled at 280 nm; a more than 5 fold reduction in absorbance is observed at the start ofthe CAX chromatogram over the other two. Timing ofthe two-stage gradient is as indicated.

[000108] Common ion exchange salts, such as sodium or potassium chloride, provide both the cationic and anionic counter-ions necessary for CAX chromatography maintained in traditional low molarity buffers, such as Tris-HCl, HEPES, and variable pH phosphate buffers. Co-elution of both acidic and basic proteins by CAX chromatography is accomplished with a standard salt gradient where proteins elute off the column based on ionic strength. Initially a two stage gradient (0 - 15% B in 12 minutes, 15 - 50% B in 7 minutes) was optimized based on providing a uniform UN absorption across the entire chromatogram, presumably to provide even protein distribution across a targeted 25 fractions for maximal resolution. Further gradient optimization was required.

Example 2: Coupling to ID-PAGE - Second Dimension.

[000109] Orthogonal to ionic-strength, protein mass is used in the second dimension with ID-PAGE to further resolve the complex brain lysate. A fraction volume of 1 mL, practical with the CAX flowrate and the BioFrac fraction collector, generally encompassed elution of entire proteins with CAX half-height peak widths generally in the order of 0.25 mL. A foreseen difficulty of CAX common with other fractionation strategies is that proteins can break across two fractions, fortunately this statistically is less likely at lower concentration when otherwise the problem would be most dramatic. Another complication is that the fraction volume is large relative to the loading volume of a gel. Microtube centrifugal filters were used to concentrate fractions to a manageable volume. A mass cutoff of 30 kDa was selected based on its association with relative pore size and not mass. Proteins > 5 kDa are routinely retained with this filter, while the larger pore size provides more rapid processing than similar 10 or 3 kDa filters. To meet the gel loading volume requirements, retentate was brought close to dryness to allow the addition of 20 μl of 2X Laemmli sample buffer, which more effectively resolubilize protein than Tris-HCl buffer, while maintaining a small volume (reproducibly 35 μl ± 5 μl). On occasion, a random fraction would take longer to run through the filter, potentially due to manufacturing variability in the membrane pore size; though no obvious effect on protein retention was observed.

[000110] Gels were visualized with traditional Coomassie Blue R250 (Figure 2), an inexpensive, easy to use stain with fixing conditions and a detection limit inline with subsequent data-dependant MSMS analysis. Stains with greater sensitivity, such as silver and Sypro Ruby, were also used successfully prior to Coomassie staining for detection of less abundant proteins.

[000111] Figure 2 revealed that the initial CAX gradient profile, based on UN absorption at 280 nm, resulted in uneven protein distribution. Protein density was particularly dense toward the end fractions leading to significant band overlap. Based on the protein distribution in Figure 2, an optimized gradient was generated for differential analysis using a 3 step slope that effectively distributed proteins throughout the available fraction space. Figure 2 shows rat cerebellum proteome visualized on ID-PAGE following CAX fractionation. 1 mg of rat cerebellum brain lysate was divided into 25 CAX fractions each resolved further by lD-polyacrylamide gel electrophoresis (10-20% acrylamide). Protein bands were then visualized by Coomassie blue staining. MKR indicates molecular weight marker lanes.

[000112] Gel-to-gel reproducibility is a potential problem for all types of protein separations involving PAGE, and a major limitation with differential analysis using 20- PAGE. CAX-PAGE reproducibility was evaluated after triplicate runs ofthe same sample. Sequential chromatograms shown in Figure 3a overlap without significant deviation. Three fraction groups evenly spaced at the begimiing (#1, 4, and 7), middle (#10, 13 and 16), and end ofthe separation (#18, 20 and 24) were loaded in triplicate onto ID-PAGE (Figure 3b). Identical protein compliments and an average intensity coπelation of 94% (Phoretix ID software) were observed. The slight variation is primarily attributed to variability in protein recovery from the ultrafiltration and gel loading. This experiment was repeated showing similar run-to-run reproducibility but with a non-uniform shift in retention time when compared with that in Figure 3. Peak shifting typical of column chromatography occurs as a combination of environmental, buffer, and column aging changes. Samples should be compared running one after another to avoid daily variations.

[000113] Figure 3 shows the reproducibility of CAX-PAGE protein separations. Figure 3 A shows a chromatogram of rat cerebellum brain tissue lysate (1 mg protein) run sequentially in triplicate by CAX. Figure 3B shows selected fractions (paired as indicated) from the three replicate CAX runs resolved and visualized side-by-side on ID-PAGE. Protein compliment remained constant while band intensity varied on average by only 6%. Example 3: CAX-PAGE Protein Recovery and Retention.

[000114] Ion-exchange chromatography has a high loading capacity, making it advantageous as a first dimensional separation. Capacity affords the ability to load a significant amount of protein permitting reasonable sample loss common in multi step processes. Of concern when combining SAX and SCX phases was the possibility of exacerbated protein loss. Protein assay results suggested an increase in protein recovery with CAX at 67% of total protein compared with 49 % for separate SAX and 59% for SCX. All assays performed were nonrialized using a fixed volume ofthe initial sample; however, fraction composition would differ between separations thereby effecting relative quantitation between ion-exchange modes. In contrast, peak area analysis indicated greater recovery from SCX at an area of 15.5 than for CAX at 12.8 and SAX at 9.25. Both methods suggested SAX may iπeversibly trap more than SCX, but no additional sample loss is observed with CAX chromatography. Roughly 60% ±10% ofthe total protein load is recovered, which is reasonable considering the multiple steps required, in particular the ultrafiltration concentration step which is known to exhibit some protein loss. Figure 4 shows the retention and recovery of CAX-PAGE separation. The results are from a chromatogram of rat cerebellum tissue lysate (750 μg) performed with SCX, SAX, and CAX with two step elution processes.

[000115] More telling from this experiment is the increased percentage of protein retained on-column using CAX chromatography. As discussed earlier, a bi-phasic medium will bind positively and negatively charged proteins more efficiently thus increasing bulk retention. So far, 88% of recovered protein is retained by CAX for gradient elution in comparison with 66% for SAX and 47% for SCX as determined by protein assay. The peak area values are similar, with CAX having the largest retained peak area of 10 (84% of total area) compared with 5.2 (55%) for SAX and 5.7 (37%) for SCX. Increased retention, the motivation for CAX, affords the ability to evenly distribute complex protein mixtures across an expandable number of fractions based on gradient optimization.

Example 4: Differential Expression Analysis.

[000116] The potential of CAX-PAGE is realized with its ability to provide differential expression maps for subsequent targeted differential RPLC-MSMS analysis. As a test case, proteomic differences between cerebellum and cortex regions of rat brain were explored. It was expected that the compliment of proteins would be similar in both tissues, but that expression would differ. Clear chromatographic differences in Figure 5a are observed between cerebellum and cortex lysates sequentially separated by CAX. For differential analysis, fractions from each run are paired and run side-by-side on ID-PAGE (Figure 5b), whereby problems of gel-to-gel reproducibility are avoided by always comparing matching fractions on the same gel. Side-by-side fraction pairing as in Figure 5b allows for direct visualization of differential expression using simple, cost efficient, visible stains (e.g., Coomassie Blue, silver, Deep Purple). Fluorescent stains such as Sypro Ruby also work well, though they require a more expensive fluorescence scanner (three times the cost for the liquid chromatography station). Whether visible or fluorescent stains are used, images are easily assessed with the Phoretix ID software. Automatic processing is performed to identify gel lanes, providing a clear boundary along the x-axis. Band height is also distinguishable, though fainter bands tend to require manual verification. Band intensity is automatically calculated along with band mass based on calibration with a traditional protein marker. Data is then output to an excel spreadsheet with adjacent bands lined up between lanes. A threshold set at 100% difference in band intensity is applied to generate a list of target bands for further analysis, thereby minimizing mass spectrometry workload in comparison with shotgun proteomic protocols.

[000117] Figure 5 shows a comparison of rat cerebellum and cortex proteomes via sequential CAX and side-by-side ID-PAGE. Figure 5 A is an overlay of cerebellum and cortex CAX chromatograms at 280 nm. (b) Side-by-side (M = cerebellum on left, X = cortex on right) pairing of 25 fractions run on ID-PAGE. Boxed bands were excised for protein identification - note letter labeling for coπelation with Tables 1 and 2.

Example 5: CAX-PAGE Differential Colorization.

[000118] A false-colorization scheme can also be used to aide manual inspection of differential expression, creating images (Figure 6) similar to those produced with 2D-DIGE (Figure 7a). The colorized image was generated by converting adjacent cortex and cerebellum lanes into green and red respectively and superimposing the two. A difference in color contrast was not of issue since both colors where generated from the same original grayscale image. Distortion between adjacent lanes was coπected with the rotation and skewing features of Adobe Photoshop to superimpose bands as best as possible. Green represented greater expression in cortex while red emphasized cerebellum. The human eye is adept at recognizing slight color shift (away from yellow at equal expression) more so than recognizing slight changes in grey band intensity. The colorization map was generally used to aide manual confirmation ofthe Phoretix ID output as it helped in distinguishing overlapping bands. [000119] Figure 6 shows a colorized rat cerebellum-cortex differential proteome display after CAX-PAGE. The colorized display was performed by overlaying adjacent lanes from Figure 5B false colored red for cerebellum and green for cortex. Color aides in manual inspection ofthe differential separation.

[000120] Figure 7 shows the rat cerebellum-cortex differential proteome display using 2D- DIGE. 2D-DIGE display ofthe pooled cerebellum and cortex lysates used with CAX-PAGE. Figure 7A is a false-color overlay of cortex Cy3 (green) and cerebellum Cy5 (red) labeled DIGE images. Figure 7B show the results of 2D differential software analysis comparing cortex and cerebellum tissue. Spots with 100% difference between samples are indicated by yellow for greater in cortex and green for greater in cerebellum, while blue indicates spots found only in one sample.

Example 6: Comparing Differential Analysis by CAX-PAGE and 2D-DIGE. [000121] Analysis ofthe same cortex and cerebellum tissue lysates was performed by 2D- DIGE a prominent alternative method that serves as a reference in determining CAX-PAGE effectiveness for differential analysis. The Cy3 and Cy5 images shown overlaid in Figure 7A were compared using Phoretix 2D image analysis software with the result illustrated in Figure 7B. Using 2D-DIGE, 45 spots were discerned as more than twice as prominent in cerebellum and 37 spots were more than twice as prominent in cortex (Figure 7B) for a total of 82 differential protein targets. In comparison, CAX-PAGE revealed 105 band intensities more than twice as prominent in cerebellum and 41 bands more than twice as prominent in cortex for a total of 146 targets, 78% more than from 2D-DIGE. Proteins of high concentration, which appeared as very large spots with 2D-DIGE also posed a problem for CAX-PAGE. As peak width in CAX chromatography is proportional to protein concentration, highly expressed proteins fell across multiple fractions appearing as dark streaks. RPLC-MSMS analysis confirmed the same protein in each streaked band. Fortunately, few of these streaks are observed with brain tissue lysate. Nine ofthe 146 band pairs were found to be redundant, reducing total targets to 137, 67% over that observed in 2D-DIGE.

[000122] CAX-PAGE provided an increased mass range for differential analysis. Ofthe 137 differential targets found with CAX-PAGE, 13 were at a mass of 100 kDa or greater. In comparison, no differential targets were uncovered above 100 kDa using 2D-DIGE. The ability to discern differences at high mass is particularly relevant in brain injury paradigms. Cytoskeletal proteins are often of great mass. This protein class is particularly prone to proteolysis associated with neuronal death after brain injury. Thereby exclusion of high mass proteins would unduly eliminate key biomarkers of brain injury.

[000123] Signal intensity differed somewhat between the two cyanine dyes giving a bias toward green or red from one gel to the next. As well, more background was detected at the emission wavelength for Cy3 over Cy5, making fainter spots more difficult to discern. Issues of lower than expected sensitivity, migration differences between labeled and unlabeled protein, and handling complexity are well known for 2D-DIGE, though the introduction of saturation 2D-DIGE labels has helped with some of these issues. [000124] In summary, CAX-PAGE generated more differential targets using less expensive more easy to use Coomassie staining and imaging. As many as 13 differential proteins were observed at a mass greater than 100 kDa using CAX-PAGE compared with none for 2D-DIGE. CAX-PAGE also maintains spatial separation of samples, permitting further differential quantitative and qualitative analysis by subsequent RPLC-MSMS.

Example 6: Resolving Power of CAX-PAGE.

[000125] Though CAX-PAGE revealed more differential targets than 2D-DIGE using the same samples, the resolving power of CAX-PAGE as performed here is lower than that of 2D-DIGE and other 2D techniques. The most common means for comparing multidimensional separations is the use of theoretical peak capacity (r-_c). Total r-_c is essentially the product of individual peak capacities for each dimension of separation. This generally assumes in the case of serial separations, that n_c for the first dimension is unaltered by the second separation. For 2D-PAGE in particular, this is not the case since protein spot diffusion occurs in two dimensions after the J-PG strip is transfeπed to SDS-PAGE. Total n_c can still be determined based on final spot dimensions (x and y axis width values) divided into the length of separation for each axis. From the 2D-DIGE separation shown in Figure 7, x-axis n_c (mainly IEF) came to be 73.5 and y-axis (SDS-PAGE) r^ 74.0. This generates a theoretical total n_c of 5440, about average for 2D-PAGE.

[000126] CAX-PAGE has an x-axis n_c equal to the number of ion-exchange fractions collected, in this case 25 or a third that of IEF, but independent of x-axis band broadening on ID-PAGE. In contrast, CAX-PAGE has twice the peak capacity along the y-axis at 143, achieved as a result ofthe larger x-axis width and the stacking gel at the top ofthe ID-PAGE (not used with 2D-P AGE). Despite a shorter gel length, the greater y-axis ri_c of CAX-PAGE partially compensates for the small fraction number producing a total ri_c of 3570, which is 34.4% shy of that calculated for 2D-DIGE. Peak capacity can also be stated as a working value, which in this case was calculated to include a rectangular separation space beyond which no proteins migrate. Determined from the images shown in Figures 5 and 7, the working values for CAX-PAGE and 2D-DIGE are 3120 and 4030 respectively, indicating that the actual resolving power of these techniques are even closer. To improve CAX-PAGE, we recently moved to larger format Protean II Bio-Rad gels (16 cm x 16 cm), which come prefabricated with up to 20 wells. Preliminary results show an increased y-axis n_c of 211, and along with an expansion of CAX separations to 36 fractions (two gels per sample), provides a theoretical peak capacity of 7600.

Example 7: Differential Semi-Quantitation and Protein Identification by Capillary RPLC- MSMS — Tliird and Fourth Dimensions.

[000127] A notable advantage of CAX-PAGE, as well as the other novel differential approaches discussed in the previous section, over 2D-DIGE is the maintenance of spatial separation between each sample. This is not possible with 2D-DIGE since inherent to this technique, indeed the driving force behind it, is that samples are mixed together and run simultaneously on the same gel to avoid gel-to-gel variability. Maintaining spatial separation between samples as afforded by CAX-PAGE is essential for further differential analysis. The presented multidimensional protocol involves selection of differential targets identified after CAX-PAGE, excision of these band pairs, digestion with trypsin, and peptide separation using capillary reverse phase liquid chromatography. Separation of tryptic digests is integral to this protocol as sample complexity is further increased by in-gel digestion, and it is often the case that more than one protein is present within the excised band. Peak capacity for capillary RPLC is high due to the enhanced efficiency of small columns, with r-_c values ranging between 100 and 200. RPLC elutes tryptic peptides spread out in time onto a tandem mass spectrometer, the fourth dimension of separation using mass-to-charge. The peak capacity of a dynamic exclusion MSMS scan method can be calculated as the parent ion scan width (800 m/z) divided by the dynamic exclusion width (3 m/z) resulting in an n_c of 267. Combining the four separation dimensions provides an immense total i_c of 1.43x10⁸. Assuming a peptidome estimate of 5x10⁴ proteins divided into 100 peptides the component number (m) is 5xl0⁶. That would indicate a system saturation factor (α) of only 3.50% (α = m/n_c). At this α, 93.2% of tryptic peptides would be independently resolved by this protocol using Poisson statistics. In other words, the theoretical peak capacity for this protocol is significantly greater than the assumed component number such that nearly all components will be resolved from one another even though the working peak capacity ofthe system is considerably less than theoretical.

[000128] The power of this multi-dimensional protocol lies in the use of four independent physical properties: 1) protein charge distribution; 2) protein molecular weight; 3) tryptic peptide hydrophobicity; and 4) tryptic peptide mass-to-charge. To proceed, three assumptions were made with regard to the MSMS data. The first was that having been visible with Coomassie staining, a protein would be identified by two or more peptides using strict cross-coπelation values, since the detection limit of Coomassie stain and dynamic exclusion MSMS are similar with our instrument. The second assumption is that in order to have produced a band intensity difference of 100% or more between samples the responsible protein would have had a similar or greater expression level relative to background proteins. The last assumption is that only the differentially expressed protein, not equally expressed background proteins, will exhibit a discernable difference in the number of peptides identified between the two samples, taking advantage ofthe semiquantitative nature of peptide number in bottom-up MSMS analysis.

[000129] To evaluate the protocol two protein groups were selected for RPLC-MSMS analysis: (1) a random selection of band pairs from the CAX-PAGE differential target list (those showing a 100% difference in band intensity) as indicated in Table 1; (2) a random selection of band pairs that did not make the differential target list (less than 100% difference in band intensity) as indicated in Table 2. In total, 85% of MSMS runs fulfilled the first assumption iπespective of whether the band was differential or not. The fact that 15% of bands did not reveal obvious identified proteins is not surprising considering that most of these bands tended to be of low intensity. An enhancement in mass spectrometer sensitivity, possible with the new generation of linear ion traps, would improve protein identification determination.

[000130] To assess the validity of assumptions two and three, we compared the two groups (Table 1 and 2) as to how often the semi-quantitative result using peptide number either matched, did not match, or was inconclusive when compared to CAX-PAGE band intensity. In the first group, both peptide number and band intensity reflected higher expression in the same tissue 89% ofthe time. An inconclusive determination from peptide number happened only 7% ofthe time, with only 1 case (4%) where the two results clearly did not match. The success rate of 89% was in stark contrast with results from group 2, where only 28% of cases resulted in a match ofthe same tissue. Without a 100% difference in band intensity, the peptide results were just as likely to predict a mismatch (also 28%) in tissue assignment. In 44% of cases a clear difference in peptide number could not be discerned, simply because the identified protein was expressed near equally in both samples. In brief, using the two step differential analysis ofthe CAX-PAGE/RPLC-MSMS platform will result 76% ofthe time in a verified definition of a particular protein being expressed more than twice as much in one sample over the other. This value can be improved by using a more sensitive mass spectrometer that would more conclusively identify peptides thereby improving semi- quantitative evaluation and protein identification.

[000131] Surnmarizing the differential findings, differentially identified proteins in Table 1 fit into three distinct protein classes known to be prominent in the brain and listed here in order of prevalence: metabolic enzymes such as alpha enolase, pyruvate kinase 3, transketolase, GMP synthase, fatty acid synthase, etc.; neuronal function proteins such as albumin, calbindin 1 & 2, translin, transferrin, etc.; microtubule proteins such as chloride intracelmlar channel 4 and MAP2. Proteins were identified over a wide molecular weight distribution from 16 to 273 kDa. This is a notable improvement over 2D-PAGE, which under represents proteins above 120 kDa, and far exceeds the cuπent mass range of top-down mass spectrometry approaches. Another advantage of CAX-PAGE is that hydrophobic membrane proteins are readily soluble in the employed SDS lysis buffer, and should not precipitate out during separation, a known problem with 2D-PAGE. This however was not confirmed here likely because membrane proteins are generally of low concentration and only 53 bands were excised.

[000132] Table 1. Semi-quantitative results and protein identification of gel band pairs showing greater than 100% difference in intensity between cerebellum and cortex - differential target list.

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[000134] CAX-PAGE also can include use of an ion-exchange columns with a smaller i.d. to provide an increase in column efficiency and a reduction in fraction size comparable to what can be loaded onto commercial ID large format gels. This would make CAX-PAGE automation more comparable with liquid phase 2D techniques that use fraction collection between dimensions without further processing. CAX-PAGE immobilizes protein within a gel matrix and affords a convenient means of visible detection with the considerable resolving power offered by ID-PAGE. High throughput staining, robotic band excision and digestion will add to largescale uses. With robotic digestion, samples are automatically placed into 96 well plates that interface directly with an autosampler for capillary RPLC- MSMS, which itself is automated for data acquisition and database searching. [000135] Preliminary experiments with differential analysis between three different stroke injury conditions have been successful. The platform using the same fraction from each sample is grouped on a single gel (i.e., fraction 1 from each sample on gel 1 etc.). The upper limit to the number of samples is therefore determined by the number of lanes within a single gel (up to 19 samples with large format gels). CAX-PAGE/RPLC-MSMS in comparison with other separations strategies does not provide a direct evaluation of isoelectric point (pi). This bit of information is particularly useful when spot location on a 2D map is employed for protein identification as often done with 2D-PAGE; however, pi as determined by isoelectric focusing, free-flow electrophoresis or chromatofocusing is also an additional means to confirm protein identity as determined by mass spectrometry methods. 2D chromatography employing these separations on the other hand does not predict protein mass. Instead unpredictable hydrophobicity is used, which forgoes use of 2D map databases for protein identification. Protein mass is used with CAX-PAGE as a secondary confirmation of protein identity. Additionally, a preliminary investigation showed a correlation between CAX elution and pi. Though the precision of this indirect relationship is low, foreseeably, CAX fraction could be used for validation purposes even if not for assigning pi. In application of CAX-PAGE, mass validation is performed in light of possible protein degradation, common after brain injury.

[000136] Differential protein expression analysis allows scientists to map relevant cellular or tissue changes in response to development, environmental stimulus, injury, or disease. The complexity of biological samples has driven the means to resolve and quantify the resulting proteome by use of high resolution separation techniques. A novel 4D approach was presented based on combining bi-polarity ion exchange chromatography in tandem with gel electrophoresis for protein separations followed by capillary reverse phase liquid chromatography online with tandem mass spectrometry for targeted peptide analysis, termed CAX-PAGE/RPLC-MSMS, with a combined theoretical peak capacity of 1.43x10⁸. Straightforward to perform, the platform utilizes traditional visualization stains for cost savings and two complimentary differential determination strategies for validation. The platform was demonstrated for differential analysis between cerebellum and cortex tissues, a test model for biomarker discovery in brain. Using protein separations, 137 distinct targets were revealed out of which 13 had a mass greater than 100 kDa. rom the 137 targets, 33 were randomly selected for further peptide analysis by capillary RPLC-MS/MS. Differential expression was confirmed and protein identification was determined in 76% and 85% of cases, respectively. Future efforts are focused on improving chromatographic efficiency for direct coupling with larger format ID-PAGE. The platform is currently being applied to biomarker discovery for clinical diagnostics of traumatic brain injury, stroke and substance abuse.

Other Embodiments [000137] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope ofthe invention, which is defined by the scope ofthe appended claims. Other aspects, advantages, and modifications are within the scope ofthe following claims.

Claims

What is claimed is: 1. A method of isolating, quantifying and identifying the biomarker associated peptides, comprising: obtaining a crude biological sample(s); clarifying the sample(s) via centrifugation and ultrafiltration; subjecting the samples sequentially to bi-phasic ion-exchange chromatography and obtaining fractions; separating fractions by polyacrylamide gel electrophoresis into bands according to molecular weight and quantitatively imaging band density and evaluating protein expression; cutting selected bands from the polyacrylamide gel and subjecting them to in-gel digestion; subjecting the digested bands to capillary reverse phase liquid chromatography in tandem with mass spectrometry; thereby, isolating, quantifying and identifying the biomarker associated peptides.

2. The method of claim 1, wherein bi-phasic ion-exchange chromatography comprises at least a plurality of gradients.

3. The method of claim 1, wherein the bi-phasic ion exchange chromatography comprises at least a two step gradient.

4. The method of claim 1, wherein the bi-phasic ion exchange chromatography comprises a three step gradient.

5. The method of claim 1, wherein the bi-phasic ion exchange chromatography comprises a five step gradient.

6. The method of claim 1, wherein the bi-phasic ion exchange chromatography comprises a ten step gradient.

7. The method of claim 1, wherein the bi-phasic ion exchange chromatography comprises between about a two step gradient up to a twenty step gradient.

8. The method of claim 2, wherein the two-step gradient comprises a linear transition from 0% to about 15% in a volume of about 12 mL up to 50 mL.

9. The method of claim 3, wherein the three-step gradient comprises a linear transition from about 15% to about 50% in a volume of about 7 mL up to 50 mL, held at about 50% in a volume of about 2 mL up to 50 mL and re-equilibrated to 0% in about 1 mL up to 50 mL volume.

10. The method of claim 1, wherein the bi-phasic ion exchange chromatography comprises a plurality of ion-exchange media.

11. The method of claim 10, wherein the ion-exchange media comprise weak anion and cation exchangers mixed with strong anion and cation exchangers.

12. The method of claim 1, wherein the fractions obtained from the bi-phasic ion- exchange chromatography are concentrated prior to polyacrylamide gel electrophoresis.

13. The method of claim 1 , wherein the polyacrylamide gel comprises a gradient of between about 1% up to 50%.

14. The method of claim 1, wherein the polyacrylamide gel comprises a gradient of between about 4% to about 20%

15. The method of claim 1 , wherein the polyacrylamide gel is visualized by gel stains.

16. The method of claim 1 , wherein bands of proteins and peptides separated on SDS-PAGE gels are quantified by densitometric measurement.

17. The method of claim 16, wherein differentially expressed bands are quantified by densitometric analysis.

18. The method of claim 1, wherein the excised bands are subjected to enzymatic digestion.

19. The method of claim 18, wherein the enzyme digested bands are subjected to reverse phase liquid chromatography.

20. The method of claim 1, wherein ric values ofthe reverse phase liquid chromatography are between about 100 to about 250.

21. The method of claim 1 , wherein fractions eluted from the reverse phase liquid chromatography directly flow into the mass spectrometry and separated by mass-to-charge.

22. The method of claim 1, wherein n_c values are at least about 1 x 10⁵.

23. The method of claim 1, wherein the n_c values are about 1 x 10⁶.

24. The method of claim 1, wherein n_c values are about 1 x 10⁷.

25. The method of claim 1, wherein n_c values are about 1 x 10⁸.

26. The method of claim 1, wherein n₀ values are about 1 x 10⁹.

27. The method of claim 1, wherein n_c values are about 1 x 10¹⁰.

28. A method of isolating, quantifying and identifying proteins and/or peptides in complex biological mixtures, said method comprising: obtaining a crude biological sample(s); clarifying the sample(s) via centrifugation and ultrafiltration; subjecting the samples sequentially to bi-phasic ion-exchange chromatography and obtaining fractions; separating fractions by polyacrylamide gel electrophoresis into bands according to molecular weight and quantitatively imaging band density and evaluating protein expression; cutting selected bands from the polyacrylamide gel and subjecting them to in- gel digestion; subjecting the digested bands to capillary reverse phase liquid chromatography in tandem with mass spectrometry; thereby, isolating, quantifying and identifying the proteins and/or peptides.

29. The method of claim 28, wherein bi-phasic ion ion-exchange chromatography comprises at least a plurality of gradients.

30. The method of claim 28, wherein the bi-phasic ion exchange cliromatography comprises at least a two step gradient.

31. The method of claim 28, wherein the bi-phasic ion exchange cliromatography comprises a three step gradient.

32. The method of claim 28, wherein the bi-phasic ion exchange chromatography comprises a five step gradient.

33. The method of claim 28, wherein the bi-phasic ion exchange chromatography comprises a ten step gradient.

34. The method of claim 28, wherein the bi-phasic ion exchange chromatography comprises between about a two step gradient up to a twenty step gradient.

35. The method of claim 28, wherein the bi-phasic ion exchange chromatography comprises a plurality of ion-exchange media.

36. The method of claim 35, wherein the ion-exchange media comprise weak anion and cation exchangers mixed with strong anion and cation exchangers.

37. The method of claim 28, wherein the fractions obtained from the bi-phasic ion-exchange cliromatography are concentrated prior to polyacrylamide gel electrophoresis.

38. The method of claim 28, wherein the polyacrylamide gel comprises a gradient of between about 1% up to 50%.

39. The method of claim 28, wherein the polyacrylamide gel comprises a gradient of between about 4% to about 20%

40. The method of claim 30, wherein the two-step gradient comprises a linear transition from 0% to about 15% in a volume of about 12 mL up to 50 mL.

41. The method of claim 31 , wherein the three-step gradient comprises a linear transition from about 15% to about 50% in a volume of about 7 mL up to 50 mL, held at about 50% in a volume of about 2 mL up to 50 mL and re-equilibrated to 0% in about 1 mL up to 50 mL volume.

42. The method of claim 28, wherein the polyacrylamide gel is visualized by gel stains.

43. The method of claim 24, wherein bands of proteins and peptides separated on SDS-PAGE gels are quantified by densitometric measurement.

44. The method of claim 43, wherein differentially expressed bands are quantified by densitometric analysis.

45. The method of claim 28, wherein the excised bands are subjected to enzymatic digestion.

46. The method of claim 45, wherein the enzyme digested bands are subjected to reverse phase liquid chromatography.

47. The method of claim 28, wherein n_c values ofthe reverse phase liquid chromatography are between about 100 to about 250.

48. The method of claim 28, wherein fractions eluted from the reverse phase liquid chromatography directly flow into the mass spectrometry and separated by mass-to- charge.

49. The method of claim 28, wherein ri_c values are at least about 1 x 10⁵.

50. The method of claim 28, wherein the ri_c values are about 1 x 10⁶. n

51. The method of claim 28, wherein n_c values are about 1 x 10 .

52. The method of claim 28, wherein n_c values are about 1 x 10⁸.

53. The method of claim 28, wherein n_c values are about 1 x 10⁹.

54. The method of claim 28, wherein n_c values are about 1 x 10¹⁰.

55. A method of isolating and differential quantitative analysis of proteins and/or peptides in complex biological mixtures, said method comprising: obtaining a crude biological sample; subjecting the sample to a bi-phasic ion-exchange chromatography and obtaining fractions; running the fractions obtained in order of elution side-by-side on a polyacrylamide gel electrophoresis allowing for differential comparison; quantifying bands obtained by polyacrylamide gel electrophoresis by densitometric scanning; selecting bands which are differentially expressed at least about two-fold as compared to a normal control; digesting the differentially expressed bands with enzyme; subjecting the enzyme digested bands to capillary reverse phase liquid chromatography online in tandem with mass spectrometry; thereby, isolating and quantifying the isolated proteins and/or peptides.

56. The method of claim 55, wherein differential expression of bands on the polyacrylamide gel are validated by comparing peptide quantity difference with gel band density differences.

57. The method of claim 55, wherein bi-phasic ion-exchange chromatography comprises at least a plurality of gradients.

58. The method of claim 55, wherein the bi-phasic ion exchange chromatography comprises at least a two step gradient.

59. The method of claim 55, wherein the bi-phasic ion exchange cliromatography comprises a three step gradient.

60. The method of claim 55, wherein the bi-phasic ion exchange chromatography comprises a five step gradient.

61. The method of claim 55, wherein the bi-phasic ion exchange chromatography comprises a ten step gradient.

62. The method of claim 55, wherein the bi-phasic ion exchange chromatography comprises between about a two step gradient up to a twenty step gradient.

63. The method of claim 55, wherein the bi-phasic ion exchange cliromatography comprises a plurality of ion-exchange media.

64. The method of claim 55, wherein the ion-exchange media comprise weak anion and cation exchangers mixed with strong anion and cation exchangers.

65. The method of claim 55, wherein the fractions obtained from the bi-phasic ion-exchange chromatography are concentrated prior to polyacrylamide gel electrophoresis.

66. The method of claim 55, wherein the polyacrylamide gel comprises a gradient of between about 1% up to 50%.

67. The method of claim 55, wherein the polyacrylamide gel comprises a gradient of between about 4% to about 20%

68. The method of claim 55, wherein the polyacrylamide gel is visualized by gel stains.

69. The method of claim 55, wherein the bands are digested by enzymes selected from the group consisting of hydrolases, esterases, carbohydrases, nucleases, deaminases, amidases, proteases, hydrases, fumarase, enolase, aconitase carbonic anhydrase, oxidases, dehydrogenases; transglycosidases; transphosphorylases phosphomutases, transaminases; transmethylases, transacetylases, desmolases, isomerases; and ligases.

70. The method of claim 69, wherein the enzyme is a tryptase.

71. The method of claim 55, wherein the enzyme digested bands are subjected to reverse phase liquid chromatography.

72. The method of claim 55, wherein ri_c values of the reverse phase liquid chromatography are between about 100 to about 250.

73. The method of claim 55, wherein fractions eluted from the reverse phase liquid chromatography directly flow into the mass spectrometry and separated by mass-to- charge.

74. The method of claim 55, wherein li_e values are at least about 1 x 10⁵.

75. The method of claim 55, wherein the l _e values are about 1 10

76. The method of claim 55, wherein r-_c values are about 1 x 10⁷.

77. The method of claim 55, wherein n_c values are about 1 x 10 .

78. The method of claim 55, wherein n_c values are about 1 x 10⁹.

79. The method of claim 55, wherein ri_c values are about 1 x 10¹⁰.