| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Pathology and Center for Immunology, Washington University School of Medicine, and
Department of Chemistry, Washington University, St. Louis, MO 63110
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In our analysis of HEL peptides, we are concerned with the identification of those that appear to be represented to a much lesser extent than 48–62. What is the nature of these peptides, and what are their reasons for being "subdominant" or "minor"? How do we go about examining them, and what are the technologic limitations for their isolation, keeping in mind that these peptides could be those with lower affinity for MHC molecules? Indeed, it is likely that a major issue in MHC-peptide analysis is the isolation of low affinity binding peptides. Mass spectrometry analysis of MHC-bound peptides has been the technique of choice since its introduction by Hunt et al. (18). Their initial studies revealed that MHC molecules contained an abundance of peptides estimated at greater than 2000 self-peptides for the class II I-Ad (18) predominantly derived from the processing of membrane and vesicular proteins (18, 19, 20). A common strategy for isolating peptides involves the separation of a heterogeneous peptide population by reverse-phase HPLC, screening each fraction with T cell hybridomas, and then identifying the peptides that trigger the T cell by mass spectrometry or Edman degradation (10, 21, 22, 23). This approach may skew the results against the weaker binding peptides for two reasons. First, each peptide may bind to APC with markedly different strengths and kinetics. Second, since each HPLC fraction consists of multiple peptides, these may compete for binding to the class II molecules on the APC. As a consequence, the T cell screen may not detect weaker binding peptides, thus favoring peptides of higher binding strength. In addition, low affinity peptides may be lost preferentially during the biochemical purification procedure.
Recently, the combined strategy of immunoaffinity chromatography and mass spectrometry analysis was employed for the characterization of peptide fragments from proteins (24, 25, 26) and for the isolation of limiting amounts of protein from complex biologic material (27, 28). We have now adapted the principle of immunoaffinity capture and developed it as a strategy to isolate low abundance class II-associated peptides. Using a monoclonal anti-peptide Ab, we recovered picomole-to-femtomole levels of HEL peptides, which were previously identified in the tryptic fragment 34–45 (29).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We used the murine B cell lymphoma line M12.C3.F6 transfected with a membrane form of HEL (M12-Ak mHEL) (11). The HEL fusion protein was engineered with site-directed mutagenesis by using a procedure previously described by Brooks et al. (30). Our control cell line, M12.C3.F6, expressing class II I-Ak (M12-Ak) (31), and M12-Ak mHEL were cultured in DMEM supplemented with 5% FCS. The M12-Ak mHEL process and present HEL peptides equally to M12-Ak given exogenous HEL (11).
Production of mAbs
mAbs to HEL peptides were produced by standard hybridoma procedures. Briefly, the synthetic HEL peptide 34–45 was coupled to the carrier protein keyhole limpet hemocyanin using bromoacetyl succinimide (Sigma, St. Louis, MO). CAF.1/J mice (The Jackson Laboratories, Bar Harbor, ME) were first injected i.p. with peptide-hemocyanin in CFA (Sigma), followed by three i.p. injections in incomplete CFA (Sigma) at 4–6-wk intervals. Finally, 3 days before B cell fusion, the mice were boosted i.v. with 25 µg of peptide-hemocyanin in sterile PBS. Splenocytes were fused with the myeloma fusion partner P3X63.Ag8 at a ratio of 5:1. Cell fusion was induced with polyethylene glycol 1500 (Boehringer Mannheim, Indianapolis, IN) by using the standard fusion protocol.
The hybridoma supernatants were screened by an ELISA assay in which 96-well Maxisorp plates (NUNC, Roskilde, Denmark) were coated with 5 µg/ml of denatured HEL (Sigma). The positive wells were then cloned. Six Abs were found to be specific for the HEL peptide 34–45, of which VAL-3 (isotype IgG1) was used. The epitope of this Ab is on the amino terminus spanning from residues 34–38 (FESNF). VAL-3 hybridoma cells were injected into pristane (Sigma)-treated SCID mice, and the IgG1 Ab was purified from ascites using protein A-Sepharose (Sigma).
Synthesis of synthetic peptides
Peptides were synthesized by F-moc chemistry (model 432A; Applied Biosystems, Foster City, CA) and purified by reverse-phase HPLC (600E; Waters, Milford, MA). The sequences of all peptides were subsequently confirmed by mass spectrometry. For binding studies, peptides were radioiodinated (125I) on Tyr residues using the chloramine-T method (32). Each peptide was iodinated to a specific activity of 0.5 mCi/1.5 nmol of peptide.
Isolation of I-Ak-associated HEL peptides
I-Ak molecules were isolated from M12-Ak
mHEL B lymphoma cells (10) and grown to a cell density of 2 x
105 cells/ml to yield a total of 1010 cells.
These cells were lysed in the presence of MEGA 8/MEGA 9 detergent
(Sigma) and enzyme inhibitors (PMSF, iodoacetamide, and leupeptin) as
described (10). The I-Ak molecules were isolated by
immunoaffinity chromatography using the anti I-Ak Ab 40F
(33). The peptides were subsequently released by acid treatment. In the
experiment shown in Fig. 1
, peptides were
separated by conventional reverse-phase HPLC (600E; Waters). Each
250-µl fraction was tested in a T cell bioassay using either the T
cell hybridoma 3A9, which is specific for the core sequence 52–61, or
A6A2, which recognizes the tryptic fragment 34–45 (29, 34). Added to
each well were 105 T cells and 5 x 104
APCs (M12-Ak) in a total volume of 200 µl. After 18
h, the supernatant was tested for the presence of IL-2 using the
IL-2-dependent cell line CTLL. The fractions with detectable levels of
IL-2 were dried down and reconstituted with a 5% acetonitrile, 0.5%
trifluoroacetic acid (TFA) solution. The fractions were then pooled and
analyzed by matrix-assisted laser desorption/ionization time of flight
(MALDI-TOF) or tandem mass spectrometry.
|
Analysis by mass spectrometry
For MALDI-TOF mass spectrometry analysis, 0.5 µl of the sample (from the standard method) was mixed with 0.5 µl of the MALDI matrix solution 4-hydroxycyanocinnamic acid (Hewlett-Packard, Chicago, IL) on the matrix plate. MALDI-TOF analysis was performed in the linear mode on a PerSeptive Voyager-RP Reflectron instrument (PerSeptive Diagnostics, Framingham, MA) using the Voyager and GRAMS/386 software. The accelerating voltage was 25–30 kV, and the ions were desorbed by an N2 laser at 1 = 337 nm with a 3-ns pulse width.
Samples from many of the experiments were subjected to capillary reverse-phase HPLC mass spectrometric analysis. Using the Rheodyne 7125 injector, we injected 15 µl of the prepared sample into the reverse-phase HPLC, which consisted of a Zorbax C18 0.3-mm x 25-cm column (MicroTech, Sunnyvale, CA). Conditions of the gradient were as follows: solvent A (6% acetic acid in water) was kept constant at 10%; solvent B (acetonitrile) was kept constant at 2% for the first 5 min, increased from 2 to 15% in the next 5 min, and then increased from 15 to 90% at a rate of 0.9%/min; and solvent C (water) maintained the mixture at 100% while the eluent flow rate was maintained at 4.5 µl/min. The HPLC pump was a Waters 600MS equipped with pulse damping (silk board). The flow from the pump was split by using the LC packing accurate splitter with a 0.3-mm-wide column calibrator (LC Packing, San Francisco, CA). The total column effluent was then directed at the mass spectrometer.
Peptides were identified by mass and sequenced on a Finnigan liquid chromatography quadropole ion-trap mass spectrometer (Finnegan, San Jose, CA) (i.e., in the MS and MS/MS modes, respectively). For MS mode, the scan range was at a mass-to-charge ratio (m/z) of 600-1300 in the profile mode, in which every three microscans were averaged to one scan. Acquisition was started 10–15 min after the commencement of the LCQ run. For MS/MS mode, the scan range was m/z 250-1850, again in profile mode, in which every three microscans were averaged to one scan. The parent ion was isolated with a mass window that was 2 m/z wide, and the collision energy was 25% of the maximum energy. Sequence analysis was performed by comparing the experimental ion mass with the calculated ion mass or by an automated protein database sequencing program (SEQUEST; John Yates, University of Washington, Seattle, WA) on an ICIS workstation (Finnegan).
Peptide-I-Ak binding strength assay
Detergent-solubilized I-Ak, purified from the T2-Ak cell line (provided by Dr. P. Cresswell, Yale University School of Medicine New Haven, CT) by affinity chromatography, was incubated with a radioactively labeled reference peptide (125I-labeled YEDYGILQINSR), a high affinity binder to I-Ak (35). The amount of peptide required to compete out 50% of this reference peptide was determined by adding known amounts of unlabeled test peptide. Each reaction contained 10 µl of test peptide at different dilutions, 0.25 pmol of 125I-reference peptide, and 25 pmol of purified I-Ak. After incubating at room temperature for 72 h, the peptide-Ak complex was separated from free peptide by spinning the material through a Bio-Spin P6 gel filtration column (Bio-Rad). The excluded material was counted using a gamma counter (Wallac, Turku, Finland). The amount of test peptide blocking 50% of the binding was standardized against the reference peptide, which was given a relative inhibitory capacity (RIC-1) of 1, where RIC-1 = Amounttest/Amountreference. Thus, a low RIC-1 value indicates a strong binding peptide to I-Ak, while a high RIC-1 indicates a weaker binder.
SDS stability of peptide I-Ak complexes
The strength of a peptide-MHC complex was also measured by its
resistance to denaturation to SDS when run in SDS-PAGE (36, 37).
Briefly, the peptides were radioactively labeled (125I) and
incubated with purified I-Ak for 48 h at room
temperature. The complex was recovered by immunoprecipitation using the
40F Ab at a final concentration of 25 µg/ml. The samples were
resolved on a 12% SDS polyacrylamide gel. As observed by
autoradiography, the peptides remaining bound to the 
ß dimer
migrated to the top of the gel (SDS-stable peptides), while the
dissociated peptides were found at the lower segment of the gel
(SDS-unstable peptides). These images were scanned by a PhosphorImager
(425E, Molecular Dynamics, Sunnyvale, CA) to determine the
relative proportion of SDS-stable complex.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies from our laboratory identified T cells directed to a tryptic fragment of HEL encompassing residues 34–45 (29). The sequence of peptide 34–45 is Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-Thr-Asn-Arg. The T cell response to this epitope in HEL-immunized mice was weaker, in comparison with the response of the dominant epitope 48–62. Moreover, peptide 34–45 was a weaker binder to I-Ak (29). Therefore, our initial impression was that the 34–45 epitope was subdominant or minor.
Using our standard method for peptide isolation, we found the natural form of the 34–45 peptide difficult to isolate from APC exposed to HEL. (The standard method consists of an analysis of peptides extracted from I-Ak by reverse-phase HPLC. Fractions that score positive in the T cell assay are then examined by mass spectrometry.) Earlier experiments that had been successful in isolating and characterizing the major epitope 48–62 failed to detect the 34–45 peptide (10). Two of three recent experiments revealed weak activity for the 34–45 HEL determinant. These results now are described so that a comparison can be made with the peptide immunoisolation method.
Class II I-Ak molecules were purified from 1010
M12-Ak mHEL cells. By acid treatment, the complex mixture
of peptides was released and then fractionated by reverse-phase HPLC.
Each fraction was screened by the T-cell hybridoma 3A9 to identify the
HEL peptides consisting of the core region 52–61. These peptides were
identified at retention times of 72 and 78 min (Fig. 1
A). In
accordance with our previous observations, analysis by MALDI-TOF mass
spectrometry revealed two prominent peptides, 48–62 and 48–63 (data
not shown); these peptides were shown to have relative yields of 32 and
56%, respectively (10, 11, 12). In addition, each HPLC fraction was
screened with the T cell hybridoma A6A2 that recognizes an epitope in
the tryptic fragment 34–45. Five fractions with a retention time of 48
min showed detectable levels of IL-2, thus providing evidence for a
second HEL determinant (Fig. 1A). It is important to
note that we required 36% (or 90 µl) of each fraction to detect
significant levels of IL-2 for this epitope, whereas only 4% (10 µl)
was required to detect the major HEL epitope 48–62.
MALDI-TOF mass spectrometry analysis of the A6A2-positive fractions
revealed no prominent peaks. Two weak signals with masses of 1814.6 and
1916.3 were comparable with the theoretical peptide masses, 1813.4 and
1914.4, corresponding to the HEL peptides 31–46 and 31–47,
respectively (Fig. 1B). Clearly, the signals of the two
peptides were too weak to incur any confidence in these masses.
We adjusted our approach by subjecting the T cell-positive fractions to
the reverse-phase HPLC online and, this time, to the electrospray mass
spectrometer, to improve the resolution of the peptide samples. Doubly
charged ions [M + 2H]2+ were observed for the dominant
HEL peptides that triggered the 3A9 T cell. Fig. 2A shows the m/z of
863 and 913, which corresponded to two members of this family, 48–62
and 47–62, respectively. In addition, the [M + 2H]2+
ions of 907.92+ and 957.72+ represented the
minor HEL peptides 31–46 and 31–47, which stimulated the A6A2 T cell
hybridoma. Ions to other minor HEL peptides were evident, such as the
1043.32+ ion (residues 31–49) (Fig. 2A).
Thus, our initial MALDI-TOF results were confirmed. However, the
abundance of these ions was still too weak among prominent
contaminating peptides and thus difficult to analyze.
|
shows a linear calibration curve of the 48–62 and 31–47 standard
synthetic peptides. By determining the area of each ion chromatogram
for the naturally processed HEL peptides 48–62 and 31–47 (Fig. 2B), the concentration of the recovered peptide was
interpolated from the calibration curve. In the same experiment, the
quantity of 48–62 peptide was 22.1 pmol compared with 1.01 pmol for
the 31–47 peptide, indicating that the display of the 31–47 peptide
was at least 22-fold lower than that of the 48–62.
|
To improve the analysis of the 31–47 HEL epitopes, we developed
an approach involving peptide-specific immunoaffinity chromatography.
Class II I-Ak molecules were purified from the lysates of
M12-Ak mHEL cells and from control M12-Ak
cells. The peptides associated with I-Ak molecules were
released by acid treatment, and HEL peptides were isolated by
immunoaffinity capture using the Ab VAL-3 directed to the 34–45
peptide. These fractions were pooled and injected into the
reverse-phase HPLC online to the electrospray mass spectrometer.
Analysis of the peptide material isolated from the B lymphoma line
expressing I-Ak showed no detectable peptides (Fig. 4A). By marked contrast,
analysis of the peptides recovered from the M12-Ak mHEL
cell line revealed an array of [M + 2H]2+ ions ranging
from a m/z of 7792+ to 1043.82+
(Fig. 4B); 13 of these ions had masses corresponding to HEL
peptides (Table I). The sequences of
these peptides were confirmed by MS/MS analysis, which was performed on
10 of 13 peptides. An example of the collision-activated dissociation
performed on the parent ions 907.22+ and
957.72+ is shown in Fig. 5.
Furthermore, a search conducted in the protein sequence database, using
the SEQUEST program, identified these sequences as HEL peptides.
|
|
|
B). In addition, we identified eight other peptides,
four of which started at position 32 (alanine) and four of which
started at position 33 (lysine). These peptides had the same variation
at the carboxy terminus as those observed with the peptides beginning
at position 31. In three experiments, we did not detect the peptides
32–48 or 33–48 (see Discussion). The shortest peptide
isolated was a 13-mer (residues 33–45) corresponding to the ion
779.02+, while the longest peptide was a 19-mer (residues
31–49) with an m/z of 1043.82+.
Analysis of the total ion chromatogram from M12-Ak mHEL
shows the elution of HEL peptides at 17.7, 18.0, and 18.2 min (Fig. 4D). These elution times are specific for the peptides
starting at position 33, 32, and 31, respectively. Based on the
m/z of each ion, the total ion chromatogram was
reconstructed for each HEL peptide (an example is provided in Fig. 4D). By contrast, the total ion chromatograms of
M12-Ak showed no corresponding HEL peptides (Fig. 4C).
Quantitation of HEL peptides extracted from I-Ak molecules
We determined the recovery of peptides during the early steps of
isolation and then attempted to quantitate them. First, the
125I-labeled HEL peptide 31–47 was incubated with purified
class II I-Ak for 48 h at room temperature. The
125I-labeled 31–47-Ak complex was separated
from free peptide and added to the cell lysate. The relative
amount of peptide recovered after each step is shown in Table II. Thus, we estimated the peptide
recovery to be about 50%.
|
We also examined the efficiency of our VAL-3 immunoaffinity column. Ten picomoles of synthetic 31–47 peptide in pH-neutralized 0.1% TFA solution was incubated with 500 µl of a 50% VAL-3-cyanogen bromide-activated Sepharose slurry (250-µl bed volume). The peptide was eluted, and the analysis was performed by MALDI-TOF mass spectrometry. A recovery of 77% was quantitated by comparing the abundance of the 31–47 peptide with our reference peptide (AAKFDSNFNTQASNRNT), which was added at 0.25 pmol/µl. Overall, the peptide recovery by the anti-peptide purification strategy before the mass spectrometry analysis was estimated to be 35%.
Finally, the naturally processed HEL peptides were recovered by
peptide-specific immunoaffinity and quantitated by electrospray mass
spectrometry. A calibration curve was generated for four
sequence-specific synthetic peptides (residues 31–47, 32–47, 33–47,
and 31–46) by injecting known amounts into the reverse-phase HPLC
online to the electrospray mass spectrometer (Fig. 6). Using the same conditions as those
for the standard peptides, we obtained ion chromatograms for each
naturally processed HEL peptide (an example is provided in Fig. 4D) and calculated the area under each chromatogram. The
recovery of each peptide was derived from the standard peptide plot
(Fig. 6).
|
). By comparison, the HEL peptide,
32–45 (4 fmol) had a 300-fold lower abundance. Interestingly, in every
case the most abundant were the peptides ending at position 46 with
asparagine, or at position 47 with threonine. The overall peptide
recovery was 4 pmol from 109 cells.
|
Our current binding studies were performed using the most abundant
HEL determinant, 31–47, instead of 34–45, which was studied
previously (29). Binding strength assays against the reference peptide
(RIC-1 (1)) revealed the HEL 31–47 peptide to be a
30-fold weaker binder (RIC-1 (30)) (Fig. 7A). By comparison, the
immunodominant HEL 48–61 peptide had an RIC-1 of 1.8,
primarily attributed to the Asp52 anchoring residue (35, 38). SDS stability is a measure of the ability of a peptide to bind
strongly to the class II ß dimer. While the immunodominant HEL
peptide 48–61 was 97% SDS stable, the minor 31–47 peptide ran
virtually 100% SDS unstable (Fig. 7B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We can state with confidence that the 31–47 peptide family consists of
truly minor peptides. The possibility that the peptides were displayed
in large amounts but lost, or were not detected by T cell assay, has
now been eliminated. There is considerable loss of peptides, as others
have noted (39, 40), when we involve a first step of HPLC separation
offline from the mass spectrometer (Figs. 1 and 2). For example, the
recovery of 31–47 was 1.01 pmol per 1010 cells (Fig. 2B) compared with the immunoaffinity capture procedure that
yielded 12 pmol (in Fig. 6, we estimated a recovery of 1.2
pmol/109 cells) (i.e., about 92% of this peptide was lost
during the initial analysis). Our estimates of occupancy of M12 C3.F6
(an APC very rich in I-Ak, about 1–2 x
106 sites per cell) with the 31–47 series peptide was
about 0.3%. In our previous studies we estimated an occupancy with the
48–62 series of 6–9% (41). In summary, the immunoaffinity step
bypasses the T cell assay, serves to concentrate the peptides, and
permits a more reliable analysis and quantitation. We believe the use
of a peptide immunoaffinity procedure will make it considerably easier
to identify the whole spectrum of MHC-selected peptides from a known
protein.
One factor that may determine the low representation of 31–47 peptides may be their affinity for I-Ak. In our analysis of HEL peptides, the most frequently displayed peptides bound to either I-Ak or I-Ek are those with high binding strength (9). Also, in the analysis of self-peptides bound to I-Ak, none is represented to the extent of 48–62, but neither do any of them display the high binding strength of 48–62. Nevertheless, more extensive analysis should tell us how strict the relationship is between extent of peptide display and binding strength for class II molecules. Concerning the 31–47 peptide, in ongoing studies we have been able to identify a major anchoring residue, Asn39, as well as residues that hinder the binding. Whether Asn39 fits into the P1 pocket of I-Ak (38) is not clear at present. Structural analysis of this peptide-I-Ak complex is in progress.
The family of 31–47 peptides, like many class II peptides, was displayed as a series varying in amino and carboxy termini. Here, the predominant species (about 53%) started at residue 31, ending at either 46 or 47. Our interpretation of the reasons for these nested peptides lies in the nature of the processing event. Our data lead us to believe that HEL is first denatured, allowing the opened molecule to interact with I-Ak molecules. I-Ak molecules select the segments of HEL bearing the higher affinity sequences. Once bound, the segment is protected from catabolism but is then trimmed by amino- and carboxypeptidases. Trimming extends to the edges of the combining site but is somewhat variable, leaving ends of different size. Our data are supported by experiments in which the addition of proline to the flanking residues of HEL-dominant peptide resulted in extensions of the peptide (11). We favor this scenario over other possibilities, such as an initial cutting of the molecule by cathepsins with subsequent selection of peptides. With this in mind, it is likely that presentation of the dominant segment, 48–62, may hinder the presentation of the 31–47 segment. We can conclude that at least 13% of the HEL molecules that donate the 31–47 epitope cannot donate the 48–62 epitope (i.e., the 13% that end up in residue 49). Future studies in which the main anchor residue of the 48–62 segment is mutated may give us information on the relationship between these two segments in binding I-Ak in vivo.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: HEL, hen egg white lysozyme; M12-Ak mHEL, M12.C3.F6 cell line transfected with a membrane form of hen egg white lysozyme; M12-Ak, M12.C3.F6 cell line expressing class II I-Ak; TFA, trifluoroacetic acid; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; m/z, mass-to-charge ratio; RIC-1, relative inhibitory capacity.
Received for publication April 10, 1998. Accepted for publication July 22, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
repressor cI protein: recognition of the same peptide presented by Ia molecules of different haplotypes. J. Immunol. 139:3973.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  J. Herzog, Y. Maekawa, T. P. Cirrito, B. S. Illian, and E. R. Unanue Activated antigen-presenting cells select and present chemically modified peptides recognized by unique CD4 T cells PNAS, May 31, 2005; 102(22): 7928 - 7933. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. W. Purcell and J. J. Gorman Immunoproteomics: Mass Spectrometry-based Methods to Study the Targets of the Immune Response Mol. Cell. Proteomics, March 1, 2004; 3(3): 193 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Lovitch, S. J. Petzold, and E. R. Unanue Cutting Edge: H-2DM Is Responsible for the Large Differences in Presentation Among Peptides Selected by I-Ak During Antigen Processing J. Immunol., September 1, 2003; 171(5): 2183 - 2186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lu, J. Vollmer, C. Moulon, H. U. Weltzien, P. Marrack, and J. Kappler Components of the Ligand for a Ni++ Reactive Human T Cell Clone J. Exp. Med., March 3, 2003; 197(5): 567 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Velazquez, I. Vidavsky, K. van der Drift, M. L. Gross, and E. R. Unanue Chemical Identification of a Low Abundance Lysozyme Peptide Family Bound to I-Ak Histocompatibility Molecules J. Biol. Chem., November 1, 2002; 277(45): 42514 - 42522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. DiPaolo and E. R. Unanue Cutting Edge: The Relative Distribution of T Cells Responding to Chemically Dominant or Minor Epitopes of Lysozyme Is Not Affected by CD40-CD40 Ligand and B7-CD28-CTLA-4 Costimulatory Pathways J. Immunol., September 15, 2002; 169(6): 2832 - 2836. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. DiPaolo and E. R. Unanue Cutting Edge: Chemical Dominance Does Not Relate to Immunodominance: Studies of the CD4+ T Cell Response to a Model Antigen J. Immunol., July 1, 2002; 169(1): 1 - 4. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Suri, I. Vidavsky, K. van der Drift, O. Kanagawa, M. L. Gross, and E. R. Unanue In APCs, the Autologous Peptides Selected by the Diabetogenic I-Ag7 Molecule Are Unique and Determined by the Amino Acid Changes in the P9 Pocket J. Immunol., February 1, 2002; 168(3): 1235 - 1243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Velazquez, R. DiPaolo, and E. R. Unanue Quantitation of Lysozyme Peptides Bound to Class II MHC Molecules Indicates Very Large Differences in Levels of Presentation J. Immunol., May 1, 2001; 166(9): 5488 - 5494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Gugasyan, C. Velazquez, I. Vidavsky, B. M. Deck, K. van der Drift, M. L. Gross, and E. R. Unanue Independent Selection by I-Ak Molecules of Two Epitopes Found in Tandem in an Extended Polypeptide Antigen J. Immunol., September 15, 2000; 165(6): 3206 - 3213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Schneider, J. Ohmen, L. Fosdick, B. Gladstone, J. Guo, A. Ametani, E. E. Sercarz, and H. Deng Cutting Edge: Introduction of an Endopeptidase Cleavage Motif into a Determinant Flanking Region of Hen Egg Lysozyme Results in Enhanced T Cell Determinant Display J. Immunol., July 1, 2000; 165(1): 20 - 23. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ma, P. E. Whiteley, P. M. Cameron, D. C. Freed, A. Pressey, S.-L. Chen, B. Garni-Wagner, C. Fang, D. M. Zaller, L. S. Wicker, et al. Role of APC in the Selection of Immunodominant T Cell Epitopes J. Immunol., December 15, 1999; 163(12): 6413 - 6423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Peterson, R. J. DiPaolo, O. Kanagawa, and E. R. Unanue Cutting Edge: Negative Selection of Immature Thymocytes by a Few Peptide-MHC Complexes: Differential Sensitivity of Immature and Mature T Cells J. Immunol., March 15, 1999; 162(6): 3117 - 3120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Unanue Chemical Features of Peptide Selection by the Class II Histocompatibility Molecules Am. J. Pathol., March 1, 1999; 154(3): 651 - 664. [Full Text] [PDF]
|
|
|
|
|
|
R. R. Latek, S. J. Petzold, and E. R. Unanue Hindering auxiliary anchors are potent modulators of peptide binding and selection by I-Ak class II molecules PNAS, October 10, 2000; 97(21): 11460 - 11465. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). The
area of the ion chromatogram was determined and plotted against each
peptide concentration. Using the same conditions, we injected 15 µl
of the 100-µl peptide sample and obtained ion chromatograms for the
naturally processed 48–62 and 31–47 peptides (shown in Fig. 2
