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Proteins (standards)

mPC-CE-UV, C-2, 50 ^m, polybrene, tITP

(62)

Proteins (aqueous humor)

mPC-CE-UV, C-2, 50 ^m, polybrene, tITP

(62)

Proteins-Bence Jones (urine)

mPC-CE-MS, SDB, 50 ^m, polybrene

(50,107)

Proteins (blood dialysate)

mPC-CE-MS, SDB, 50 ^m, polybrene

(44)

Proteins (brain dialysate)

mPC-CE-MS, SDB, 50 ^m, polybrene

(44)

Proteins (CSF)

mPC-CE-MS, SDB, 50 ^m, polybrene

(44)

Proteins (aqueous humor)

mPC-CE-MS, C-8, 50 ^m, polybrene

(50)

Proteins (aqueous humor)

mPC-CE-MS, c-2, 50 ^m, polybrene, tITP

(63)

Proteins (tears)

mPC-CE-MS, SDB, 50 ^m, polybrene, tITP

(44)

Proteins (renal dialysate)

mPC-CE-MS(MS)d, SDB, 50 ^m, polybrene, tITP

(112)

asee refs. 34, 44, for review. ^bfs, bare fused silica. "both MS and tandem MS analysis. ^Details not given. "Off-line sample loading.

asee refs. 34, 44, for review. ^bfs, bare fused silica. "both MS and tandem MS analysis. ^Details not given. "Off-line sample loading.

Fig. 5. Three dimensional plot of mPC-CE-MS analysis of an HPLC fraction from ~109 EG-7 mouse tumor cells containing peptide(s) that induced a positive T-cell stimulation response. Twenty-five microliters of the diluted HPLC fraction pressure injected and adsorbed onto an SDB membrane. Analyte elution was with ~80 nL 80:20 MeOH:H2O sandwiched between an LSB of 0.1% NH4OH and a TSB of 1% AcOH followed by separation buffer (2 mM NH4OAc:1% AcOH). Separation was carried out in bare fused silica capillary (25 ^m i.d. x 75 cm in length) at a voltage of 25 kV. MS was performed on a MAT 900 over a mass range 300-1300 amu at 3 s/decade, accelerating voltage 4.8 kV and an ESI spray voltage of 3.4 kV. ESI capillary was 200°C.

Fig. 5. Three dimensional plot of mPC-CE-MS analysis of an HPLC fraction from ~109 EG-7 mouse tumor cells containing peptide(s) that induced a positive T-cell stimulation response. Twenty-five microliters of the diluted HPLC fraction pressure injected and adsorbed onto an SDB membrane. Analyte elution was with ~80 nL 80:20 MeOH:H2O sandwiched between an LSB of 0.1% NH4OH and a TSB of 1% AcOH followed by separation buffer (2 mM NH4OAc:1% AcOH). Separation was carried out in bare fused silica capillary (25 ^m i.d. x 75 cm in length) at a voltage of 25 kV. MS was performed on a MAT 900 over a mass range 300-1300 amu at 3 s/decade, accelerating voltage 4.8 kV and an ESI spray voltage of 3.4 kV. ESI capillary was 200°C.

In a specific instance, naturally processed MHC class I peptides were isolated from ~109 EG-7 mouse tumor cells. This cell line had been transvected with the ovalbumin gene, along with the actin promoter. The cells were harvested, lysed, and MHC class I peptides obtained as described previously (108,110). Subsequently, all HPLC fractions were subjected to a T-cell stimulation immunoassay (110). Once such fraction exhibited a positive T-cell stimulation response. This fraction (~50 ^L) was then subjected to mPC-CE-MS analysis to ascertain the number and molecular weights of peptides present, and this is shown in Fig. 5. A myriad of peptide responses were detected, indicating that in most cases such analyses should be carried out in conjunction with a targeted immunoassay or other bioassay approach

Fig. 6. Membrane PC-tITP-CE-microspray-MS/MS analysis of MH22+ = 482.3. Sample obtained from another preparation of ~109 EG-7 tumor cells. Approximately 10 ^L was pressure injected onto an SDB membrane. Peptides were eluted with ~110 nL of 80% MeOH/H2O followed by a TSB of ~115 nL 0.1% NH4OH. Separation was carried out in a polybrene coated capillary (75 ^m i.d. x 80 cm) at 15 kV with reversed polarity. It was connected via a liquid junction to a capillary emitter (25 ^m i.d. x 15 mm), which was positioned ~15 mm away from the heated capillary. Separation solvent was 0.05% AcOH in 10% MeOH. Collision induced dissociation of MH22+ = 582.3 was carried out using argon as collision gas at 1 x 10-5 mbar pressure and a collision energy of 28 eV.

Fig. 6. Membrane PC-tITP-CE-microspray-MS/MS analysis of MH22+ = 482.3. Sample obtained from another preparation of ~109 EG-7 tumor cells. Approximately 10 ^L was pressure injected onto an SDB membrane. Peptides were eluted with ~110 nL of 80% MeOH/H2O followed by a TSB of ~115 nL 0.1% NH4OH. Separation was carried out in a polybrene coated capillary (75 ^m i.d. x 80 cm) at 15 kV with reversed polarity. It was connected via a liquid junction to a capillary emitter (25 ^m i.d. x 15 mm), which was positioned ~15 mm away from the heated capillary. Separation solvent was 0.05% AcOH in 10% MeOH. Collision induced dissociation of MH22+ = 582.3 was carried out using argon as collision gas at 1 x 10-5 mbar pressure and a collision energy of 28 eV.

(50). However, in this particular case, a peptide with migration time ~23 min afforded an ion MH22+ = 482.3. This corresponds to the molecular weight of an ovalbumin peptide, OVA, a known antigen of this particular mouse model system. Another EG-7 tumor cell preparation was ultimately subjected to mPC-CE-MS/MS using a polybrene-coated capillary in conjunction with microspray-MS, in order to achieve maximum sensitivity and sequence data on the peptide. The ion at m/z 482.3 was subjected to collision induced dissociation conditions previously shown to afford significant fragmentation of the precursor ion (13), and the resulting product ion spectrum is shown in Fig. 6. Based on both the clear 'y' and 'b' series of ions, as well as the returned SEAQUEST search, the sequence was determined to be SIINFEKL, confirming the presence of the OVA peptide in this fraction. This overall strategy has been used to successfully analyze and obtain other peptides sequence data from Kb EG-7, Kb EL-4, and PVG R1 rat spleen cell lines, and this is all summarized along with mPC-CE-MS conditions in Table 1.

4.3.2. Proteins from Physiological Fluids

One-step, on-line analysis of physiological fluids (e.g., urine, bile, plasma, tears) by mPC-CE-MS has substantial potential in the development of suitable biomarkers for diagnosis of disease states. We have previously reported the utilization of this approach for the detection of Bence-Jones protein(s) in urine (107). This protein(s) is a known biomarker for patients suffering from multiple myeloma. Typically, a 24-h urine sample is collected followed by numerous sample handling and separation steps in order to detect the presence of the protein. This is in stark contrast to the ~30-min analysis time of the urine sample by mPC-CE-MS. Approximately 1.5 ^L of urine obtained from a patient suffering from acute multiple myeloma was subjected to mPC-CE-MS analysis. The mass spectrum revealed a number of glycoforms of the protein at 23,680, (Parent), 23,941, and 24,144 Daltons (12,20). It is possible that the ratio of these previously undetected glycoforms may give further insight into onset and development of the disease, and this is currently under investigation.

Other demonstrations of the direct analysis of physiological fluids by mPC-CE-MS have been reported. They include blood and brain dialysate (44), cerebrospinal fluid (44), aqueous humor (50,63), tears (44), and kidney dialysate (112), and is summarized in Table 1. An analysis of physiologically of such derived fluids affords a myriad of responses, many of which are unidentified proteins. For example, aqueous humor is the extracellular fluid that fills the anterior chamber of the eye and bathes the lens, cornea, and trabecular regions. It is composed of inorganic ions, small organic molecular, and numerous peptides and proteins. It has been proposed that levels of endogenous proteins, as well as the presence of exogenous proteins may be of some diagnostic significance. In that regard we compared the mPC-CE-MS profile of aqueous humor from patients with cataracts ("normal") to patients suffering from glaucoma. This is shown in Fig. 7A and B for the base peak ion chromatogram of normal (Fig. 7A) versus glaucoma (Fig. 7B) aqueous humor. It is clear that the 'normal' aqueous humor contains many more protein responses than that of the glaucoma aqueous humor, and we are in the process of attempting to identify individual protein constituents in order to understand the comparative differences. This may give more insight into the basic mechanisms involved in the onset of glaucoma.

Fig. 7. Direct analysis by mPC-CE-MS of aqueous humor from a patient undergoing surgery for (A) cataracts, (B) glaucoma by mPC-CE-MS. One microliter of aqueous humor was pressure-injected onto a C-2 reversed phase membrane. The membrane was washed with 2 (L of 2 mM NH4OAc in 5% AcOH, and analytes were subsequently eluted with 60 nL of 80% CH3CN in H2O, followed by a TSB of 60 nL 0.5% NH4OH. Separation was carried out in a polybrene coated capillary (50 ^m i.d. x 80 cm in length) at a voltage of 15 kV. MS was performed on a MAT 900 over a mass range of 650-2200 amu at 3 s/decade. Accelerating voltage 4.7 kV and an ESI spray voltage of 3.4 kV. Instrument resolution ~500, analyte detection with PATRIC detector.

Time [min]

Fig. 7. Direct analysis by mPC-CE-MS of aqueous humor from a patient undergoing surgery for (A) cataracts, (B) glaucoma by mPC-CE-MS. One microliter of aqueous humor was pressure-injected onto a C-2 reversed phase membrane. The membrane was washed with 2 (L of 2 mM NH4OAc in 5% AcOH, and analytes were subsequently eluted with 60 nL of 80% CH3CN in H2O, followed by a TSB of 60 nL 0.5% NH4OH. Separation was carried out in a polybrene coated capillary (50 ^m i.d. x 80 cm in length) at a voltage of 15 kV. MS was performed on a MAT 900 over a mass range of 650-2200 amu at 3 s/decade. Accelerating voltage 4.7 kV and an ESI spray voltage of 3.4 kV. Instrument resolution ~500, analyte detection with PATRIC detector.

It is clear from the mPC-CE-MS analysis of aqueous humor, described earlier, that ultimately rapid identification of proteins derived from physiological fluids is necessary. This has recently been described for the identification of a renal dialysate protein (112). A number of patients at Mayo had a rare renal tumor that was shown to inhibit renal epithelial phosphate transport. Approximately 200 L of renal dialysate was collected from these patients and ultimately reduced to ~6 mL. This was achieved through a series of dialysis and size-exclusion chromatography concentration steps. The active component(s) was tracked at each stage using an opossum kidney-cell phosphate (32P-labeled) uptake assay. Approximately 100 ^L of the dialysate active fraction was assigned for mPC-CE-MS and tandem MS analyses. Initially, 20 ^L was subjected to mPC-CE-MS and one major ion response was detected at Mr = 11,729 Daltons (Fig. 8A). The remaining ~80 ^L was then digested with lysine-C proteolytic enzyme and the resulting digest subjected to mPC-CE-MS and tandem MS analysis. A series of peptides were detected including an ion at m/z 574.5, corresponding to the MH22+ = 574.5 shown in Fig. 8B. This spectrum was searched without prior interpretation using the SEAQUEST database. The resulting peptide sequence, VEHSDLSFSK was consistent with the peptide being derived from the protein ^-microglobulin.

Surprisingly, this is a common protein found in human dialysate. At present it is not clear if the elevated levels of ^-microglobulin interfere with phosphate uptake in renal epithelial cells or perhaps another minor component co-migrates with it. This problem is still under active investigation. Nevertheless, it is clear that mPC-CE-MS and tandem MS can play a significant role in rapidly and unequivocally identifying proteins.

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