9 The ,Potential of I~Jigh

Resolution Protein Mapping as A Method of Monitoring the l'iuman Immune System

N. L. Anderson and N. G. Anderson

Jdolcel/lar Anatomy fragrant, Division 0/ Biological and h1ctiica/ Research, Argonne National Laboratory,

'A rgolllle, Illinois

INTRODUCTION

Immullology traditionally deals with complex cellular systems and hetero­geneolls mixtures of cfTcctor molecules (primarily antibodies), Some sense has emerged from this cll aos through t he use of fUllct iOllal assays (ce II proliferation, killing, ete.,- or antigen-antibody reactiolls). Such an ap­proach howcvcr natll[;J1ly leaves a great deal lllH.liscovcrcd since tlte assays arc simple and tlle ~lss;lyed ohjects are complex. III this chapter we describe some experimental approaches to inllllllIlological problems using high-resolution t\\'o-dimellsioll:i1 electrophoresis (O'F:lrl'ell, 1975; Ander­son and Anuerson, 1978a, b), a mcthod that can rcsovlc thousands of proteins and C~1l thus bq;in to trctlt immunological cntities at their ap­propriate levcl of complexity. I n addition, we discuss the possible applica­tion of this work to the problem of monitoring events in the individual human immune system.

MATERIALS AnD METHODS

The two-dimcl1sional electrophoretic method used here is b~scd on that of O'Farrell (1975), and consists of isoelcctric focusing in 9 rvl urea, 2S'o

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High Resolution Prolein Mllpping 1l'l

NP-40 followed by sodium dodecyl sulfate gradient gel electrophoresis. Equipment developed in this laboratory (termed the ISO-DALT system; Anderson and Anderson [1978a, b]) enables brgc numbers (,....., lOO/tby) of analyses to be performed and standarized (Anderson ano Anderson, 1979; Anderson lind IIickman, 1979). The method is ucnaturing in both dimensions and hence protein activity is generally lost upon analysis.

Lym phocytes arc prepared usi ng Ficol- Paquc graoiell Is (Pharm :lci a) and arc radioactively LIbeled by incubation witll :1:-·S methioninc in methi­onine-free (RP.tvtI 1640 medium with or without 10l}'o felal calf serulll). Serun1 immunoglobulins arc prepared using protein A Stpharosc lllicro­columns (Pearson anJ Anderson, 1980) and human urinary proteins by cstablished conccntration proccdurcs (Anderson ct al. , 1979'(1, b).

Two-Dimensional Analysis of Human Lymphocytes

The two-dimcnsional pattern of human peripheral blood lymphocytcs is shown in Figure 1. Although scveral hundred protein spots arc visible, many more nrc revealed ill longer autoradiographic cxposurcs. How can sllch data bc dealt with ill the absence of knowledgc as to the names ano fll net ions of t Ilese molecules? I n our view (A nderson and Al1th: rSOIl,

1979; Anderson et al., 1979c), the problem may best be attacked by accumulating the following sorts of information on ~111 the spots: (1) cytoplasmic localization (i.e., is the protein' located rnalnly ill the nucleus, pbsm:l mcmbranc cytoskeleton, ctc.?); (2) chcmical characteristics (i.e., is the protein phosphorybtcd or glycosylatcd?; what is its amino acill com­position?); and (3) genctic and cOlltn)) propertics (what chromosome bears the gene in question?; is this protein !specific to the lymphocytc-or one type of lymphocyte?; what othcr proteins arc coregulatec1 with this one?; what is the lifetime of this protein's mRNA?). All of this information and quite a bit marc can be obtained from cxperiments in \vhich cells or extracts from thcm arc analyzed all 2-D gels. Thc strcngth of the gc1-brlseo approach is that information 011 lnnny spots can be obtained simultane­oLisly. Once the groundwork is dOlle, interesting questions C;1I1 be poscd as to the function of selected proteins: what docs it mean, for instance, when a protein is found to be (a) unique to lymphocytes, (b) poly­morphic, (c) loeatcd mainly in the nucleus, Cd) prouuced frolll a very short-lived messenger RNA, and (e) phosphorylated only when cclls arc exposed to a tumor promoter? Obviollsly, a satisfactory cxplanation of function requires much more than this, but at least this approach provides some objective basis for choosing proteins for uet:likd study. As bio­chemistry now stands, it is generally only those protdns delectable in

138 II. Measurements or Immunologic Parameters

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to t he Ie fl (1\ ndcrson and II ickman, 1979). and SDS electrophoresis procceds uov.·m .... ards (high Illokcuhr "" 'eight at the top) .

some preconceived assay of fUllction that can be investigated. Clearly, this is unsatisfactory in view of the fact that ollly 1-2% of the human proteins (excluding immunoglobulins) have so far bcen namcd or thought of.

A systematic 2-D gcl study of lymphocytes has at Icast two applications in the area of immunc systcm monitoring. First is the obvious desirability of looking for associations bet\vccil parlicular gcnotypes and variolls immune dysfunctions. All interesting lymphocyte polymorphic system is shown in Figure 2; the protein involved is of unknown function . Since the

High Resolution Protein Mapping 139

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Figure 2. A small section from two-dimension:d sep:lr:ltions of methionine­lal.wlcu IYlllphocytes from fOllr individuals (the ~Cl'tiol\ is equivalent to part of the tipper Idl qlladrnnt of Fig. 1). Nu rnbers inJ icate PP\;jt ions of three al k 1 ic forms of an unknown protein. A, D, C, D. are mother, father, son, and lbughler, respec­tively, and show segregation of the various nlleks (From Anderson et aI., 1980c, \',:illl permi')sion of the publisher),

horizont;ll (isoelectric focusing) POSitIons of protein spots are very sensi­tive to small charge changes (gcnerally 0.1 charge). one can assume that approximately one third of the possible amino acid substitutions would lead to detectable spot shifts. So far it appears that the number of poly­morphic proteins in nucleated cells is Illuch lower th~ll woulJ be ex.pectcd from serum protein gcnetics; although this leaves few "nonhistocom­patibility" polymorphisms to study, it Inakcs the job of recognizing rare variants and new mutations much easier. A second application involves examilling the protein pattern of lymphocytes (prepared as before directly from fresh blood) as an indication of their functional status in vivo. \Ve

140 II. Measurements of Immunologic rarameters

have found evidence that stimulation with concanavalin A, exposure to certain tumor promoters, etc. causes different distinctive changes in the pattern of Ilc\\!ly synthcsized lymphocyte protcins. Thesc observations suggcst that some nongcnetic abnormalities in the patterns of lymphocytes from various indiviuuals can 1Iltimately be interpreted as indications of the functional state of these cells ill vivo.

Two-Dimensional Mapping of Serum Immunoglobulins

Figure 3 shows the antibody light chain regions of two-dimensional gels of serum Ig from a number of individual D'ALB/c mice. The patterns are extrcmely similar, sharing approximately 79-RO'J'o of the spots. The re­su It is sl igh II Y surprising in view of the notable heterogeneity of nlost antibody responscs, but tlte similarities can be regarded as the predominant ex pression of a certa i 11 set of germ-l i ne genes in "non i mmunizedH in brcd animals. Typical patterns of human Ig show much greater heterogeneity, as expected in outbred individuals, and almost no distinct light chain spots can be resolved on gels' of the type llsed in Figure 3 (7" X 7" gels). By fUllllillg I:Hi!-cr, highcr resolulion gels, some distinct spots can be secn (particui;n\y ill tilt: serulIl of Clllccr patients; Figure 4). Each snlall, well­resolved spot is likely to be the light chain product (eithcr K 01''\) of a single clone of B-cclls . The rate of increase of the ;llllount of each spot with tillll.: is probably a rl.::tsollahk measure of the rate of growth of the associ;lted c1olle, alld hence a IIlI.:;)sure or the "strength" of one particul:tr antibody rcspOIISC . Unfortullately, it would be quite dillicult to determillc what alltigens correspond 10 each of the various antihody moleculcs pro­duced; clue to genelic !Jcterogclleity, each human illlllHllle system would prnh:lbly be a unique clse . \Vhat Illay be of inlercst, however, is the abilify 10 study tIll.: r;11c or ;lppCaral1ce and dis~\ppear;\ncc of a suostanti:t1 /l\II11hcr of clnJ1:lI prudllcts ill a L~i\'ell individual (p:lliellt). This could givc a much bro~lkr picture of hUllloral illllllUIlC competence than a test of respollse to one or a ft.:w COlllillon antigens . Ultimately, it might be ros~i hk to mOll i tor a person's ex posu re to strollg all tire ns by compari [lg

the 19 light chain micropattel'lls from serul11 salllpies obUlined at, for in­stance, yearly intervals. Initial sludic'\ to examine this possibility arc now under W:ly.

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High Resolution Protein Mnpping 141

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142 II. Measurements of Immunologic Parameters

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lInrc~ol\'l:d. nllmher or (1011 :11 products (different in the two individuals) tire ncvcrthckss visibk. 1 hc~c gels welc 12" x 12" in size (almost twice as large in each lIirlll!l\sio/l as S!;lllll.lrd gels) .

Two-Dimensional Analysis of Urinary Proteins

A great variety of proll'ins is excreted ill slTl~11 [lIllOlll1tS even by norm:1l people, al1J many Jiscascs (p:nticularly those of the kiuney) lead to dramatic illcre;lses ill the excretioll of some of these or of difTerent, normally ~Ihsenl. plllypcptidcs (Anderson et aJ., 1979a, b). Sillce the kiJllcy P;\SSCS sll1~lI, IlOllserUIll proteins rather freely, it is expected that J1lall)' molecules indiclli\'c of ~l)ecific dnl11age to a \'ariety of organs can be detected in tile urine Silllply as a result of leakage from aITectcd cells. This sitll:lliull m:l"cs possible two interesting npproaches to the monitoring of imlllulle fllllctiLHl. First, it may enable accurate assessment of damage to tr~\l1spLlIlteu or~al1s caused by the host immune systcm. If rejection erisodes ;lre illdccJ clt]Llclcrizcci by a ralher abrupt risc in cell damnge in the or~~ln, thell it could be quite useful to be nblc to follow the release i

of prokins specifiC to these cells into the blood and finally the urine. ~ 1

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High Resolution Protein Mapping 143

SCCOIlU, it is possible (0 examine protcins synthesizeJ by immune cells which themselves appear in the urine. Bcnce-Jones pro!cin~ are an obvious example. Even in this case, however, two-dimcnsion~1 analysis (Figure 5) indicates that in audition to the overwhelming amollnt of the clonal light chain, there arc detectable amounts of many other abnormal molecules present.

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114 II. Measurements 01 Immunologic Parameters

CONCLUSIONS

The principal point to be made is that the complexity of the immune system (or indeed any cell or organism) is far greater than the resolving power of allY of thc cOllventional methods of studying it. Two-dilll~nsional gel electrophoresis is the first method of sufficient resolving pov, cr to be able to catalog the cells' primary functional 'parts (the proteins) Inore or less to completion, and therefore tn enable a rational search for (he most important nnd interesting parts. Il seems clear that the trlle Inolecular basis for 111any discases. rtnd abnormalities (especially those due to genetic factors) will be open (0 elucidation only when we possess such a conl­plete catalog of the plt.)leins, heavily annotated with chemical and func­tional characteristics. Almost as a by-product of this work, we expect to find a substantial, we hope di ;'gnostically useful, set of ITIolecular indica­tors of pathologic cellular states.

ACKNOWLEDGMENTS

This work is supported by the U .S. Department of Energy under c(\l\lract No. \V-31-109-ENG-3S.

REFERENCES

Anderson , N. G . <till! Amkrson. N. L.: Analytical Techniqucs for Ccll Practiolls. XXI. Two·Dirllcllsioll ;t1 An;dysis of Serulll and Tis.suc Proteins: r ~ ~rltiplc ]SO·

cicl' tric Foclising. An:" Biochcl1l ., 85:)31-3·10 (1978a). Anderson, N. I.. and Andcrson. N. G .: AII:llylical Techniques for Cell Fractions .

X X II. '1\"0- Di Illcllsio(nl A nnl >'sis of SCIUlli and Ti ... SllC Protei II '; : 1\'1 ul Liple Grildicnt-Sbh Gel Ekctrophore'\is. An:d. lliochclIl., 85: 341-35·1 (19781» .

Andl'lsoll, N. ( •. and Andason, N. L.: l\lolccular Anatomy. Behrillg .1nst. t-.lilt., 63 : 1()9·210 (1979).

Andersoll N Goo Andersoll 1': ., 1... Tolhbcll. S. L.. Hahn. II ., GiCI", F., :llld Fd\\' :lrd s. 1.: I ,n:dylicd Tcchniqllcs for Cell rr :\ctillIK XXV. Conccntl:1tioll and T\\,o-DinH:llo.,ioll :d Flectruphorctic AlJalysis of Hlll1l;\Il Urinary ProtL'ins. An :d . nioche Ill.: 95 : 48-61 (I ~)79:1) .

Anderson, N . G ., Anderson, N. L., nnd Toll :lben. S . L. : Proteins of HUl1l:1n Urillc . I. COllcr lit (:It ion a nd A n:1lysis by Two-Dilllcnsion :1I Electrophoresis. CI i 11. Chclll .. 25: 1199-1210 (197~b).

Anderson, N . L., Edwards, J. J., Giomelli, C. S., \Villard, K. E., Tolbl ·. sen, S. L, Nancc, S . 1.., Ilickl11:1Il, B. J., Taylor, J., Coulter, n., Scantlor;), A., nnd Andcrsoll, N . G .: I lif:h Resolution Two-Dimensional Electrophoretic ~f:lrpillg o[ HUIlI:ln Proleills . 111 Proceedings of Electrophoresis '79, D. Rndob, cd ., \V. ueGruYIl:I', in press (19~Oc).

Anll n Oion, N . L. :ll1d lJidl1l;\lJ. B. J.: AI1;lI}'lic;1I techniqlles for Ccll r:r;)clil)I\<; .

X X I V. fsoL' lcclric Poi nt SI:1l1cbrds for Two-Dimension:" Eleclrophoresis. A 11:1 1. Uiochclll .• 93:312-320 (1979) .

High Resolution Protein Mapping 145

O'f;Hrcll, P. H.: JIigh Rcsolution Two-Dimensional Eleclrorhorcsi~ of Proteins, J. Bio1. Chem., 25{):4007·4021 (1975).

Pcarson, T. ami Anderson. 1..: Analytical Tcchniqucs for Cell Fractions. XXVIII. Dissection of Complex Antigenic 1\1 iXlllrcs Using Monoclon:11 ,\ ntibodics nnd Two- Dimension:11 Gel I;lcclrophorcsis. Anal. n iochem. (1980) in pre\s.

ACKNOWLEDGMENTS

The sllbmitted manuscript has been autlwred by a contr:1ctor of the U.S. Government undcr conI ract No. \V-31-109-ENG-3H. Accordingly. I he U.S. Govcrnment retains a nonexclusive, roy:t1ty-free license to rllh1i\11 or rcpro­dl1ct: the published form of this contribution. or allow others to do so, for U.S. Government purposes.

D~SCUSSION

I 1;\ DDEN: I would Ii kc to comment on lhis ekg.:mt :1IH.1 futurist ic prescil tat ion. I!l acldi lion to gene ra t i Ilg a static repertoi r~ of lYIl1 phocyte st rllc ttl r:d proteins, i l seems to me tha t one of the st rOllgcst COil t riou ti ons t h~ll III ight be m~dc wi til til is tcch nology i 11 valves the dc\'cloPIllCIl t of a dyn :\In ic repcrtoire. \Ve know from the \vork of Ed\\-'a rd J 0 11I~ ~()n in Vincellt Alfrey's laboratory that there :Ire a number of Illiclear acidic proteins, which are phosphorylated within minutes of mitot:cl1 action. \\,ith us, Dr. Johnson extended this observatioll to hormolle ;lctioll. These cll;lllges occllr within millutes-so that one docsll't have lite problems of changes of structural proteins related to proliferatiol1. aIle can study protein modifIcations with phosphorylation that would indicate which olles might be regubtory proteins. \Vithin the context of T-Iymphocyte secretion, \VC know that there is a plethora of lymphokines, the identities of which arc just beginning to be perceivc<..l on a biochemical b:1sis that :1rc sccrcted during one or another fornl of activation. The analysis of secreted labded extraccllular proteins would give another reference pro­file for meaningful lymphocyte proteins which \volllJ be easily <..listin­gllished from struclu ra 1 protei ns. I th i 11 k th is technology ofTcrs great potential for analyzing lymphocytes.

ANDERSON: \Vell, you're 100 percent correct! I ncglec(cu to mention that OUf actual current experimcntal program illclllues a seriollS efTort to try to !lnJ as many chemical substancc~ as possible which have effects on the pattern of protein synthesis in leukocytes. There are several bizarre things which I hope a pharmacologist will expl;lin to me some day. Number one: It's extremely ditlicult to find compollnds which alTeet gene expression. Therc is an intinitc number of ways to kill cells, but there is

146 II. Measurements of Immunologic Parameters

almost no way to actually switch on a gene in them. If you think about it, there should be compounds which nlake every cell in your body turn red, because they acciJentally turn on the hemoglobin genes-one of the globin genes. Apparclltly, there aren't.

Now there is, of course, the case of DMSO and erythroleukemia cells. There are all kinds of ways of making a cell fall over a difTerentiation~l event that it was about to do anyway. nut thert are very few ways of actually getting ill and pulling a wrench in the mechanisms of gene regulation nnJ making a change.

The first thing we are trying to do is to make a catalogue of all of the ways that one can cause changes in gene expression; either difTerentia­tional or simply regulatory, and thell to try all possible permutations of those in an attempt to get an idea of how nlany sep~Hate mechanisms there are that are doing this work. Thc kinases arc a good one. That's why I was asking you about the phorbal esters, because those things apparently do things to proteins in lymphocytes that you just woulun't believe. Th~y make kinase stimulation Irok like a sort of trivial event.

HADDEN: If I might follow lip all my CO!1llllcnt; \vith the 100 pcrccnt ratillg, it's h;lrd to SlOp! I ollly wish YOli WCi'C revicwing nly NIH grant rropos~l! Usillg similar tcchllologies wc ho/1l' to approach the action of the t h)' 111 i c h () r m () II C s. C () 11 sid e red wi I h i nth ~~ co n t ext or cI i ITer e n tin 1 i 011 ,

these lJorllH11lCS, of which therc arc several, nITcr the possibility indeed of pickillg out specil1c gene products relatcd to the ontogeny of T­lymphocytes.

ANDERSON: I won't go so far as to say that it can't fail, but it will certaillly work!

tv! A 0: I W~lS very m llch i III prcssed by your prescl1 tat iOll. I thought t 11 ;It

your presentation perhnps offers a br;\IH.l new approach ill this area. Did you COil sider, or dn you propose to consider, the lise of some other COil·

\'cntion;L1 inH1lllllo;lssay mcthods or will you considcr tile lise of a vcry scnsitive mcthlHJ, such as RIA, irnmullollllorcsccnce assay, or roscttc tcchn iq lie in oruer to com pa re you r work 111 atcri als and approachcs wi t h t he all t ise ra prod llccd ,lga i J1st sc rllm cOl1sti tllen ts, J ones' protein, or ~ 11 Y other kinu of protcins which may be available to you? ]f you don't have the antisera, you Cln 1l1~1kc it ill your own I~lboratory. And if you w~nt to 111;[ Ke a n ~\l1 t i sc rtlill ag;1 i 11St a chem ica 1 com pOllnd, you make the h;l ptell antibody out of it so that you can Illake a comparison study such as I melltiollcd 11HH11ClitS (lgO. I do think that it is esscntial in this case. ANDEI~SO~: Ycs. Il's absolutely essential to try to iJentify, to start

With, liS 1l1,lflY of tllese spots ;IS we call. One of those is bcta-2 micro­globulin. Therc's b:llHI across tile middlc right by actin th~lt contains I11ll:;t

of the H LA .llokcli Ics; t ha t's most of the genetic dit1erencc be t wee II

High Resolution Protein Mapping 141

peoplc. Clearly onc wants to identify all thc spots which arc known, and thosc arc the spots for which onc potentially could do radioiml1lUIlOassays, spots for which antibodies arc av~\il:!blc.

Thc point is that beta-2 microgloblliin just doesn't include very many proteins; I he second poi 11 tis t Iw tit uoesn 't necess~Hi I y i IlCllld~ t he best proteins to be looking at. It includes the proteins which arc easy to isolate intact, which is a vast minority of proteins; also it includes proteins that OIlC can gel from people who YOli believe h:l\'c prouuceJ pure antibodies or pure proteins. \Ve try to uo this to the extent that we call, but the main point is to do the analysis from an objectivc poillt of vicw, where one do'cs not start out prejudiced in favor of orllithine uec:II'boxylase, just because you read in a paper somewhere that it h:IS something to do with something! \Ve st:lrt with a spot, and \'-t'ltel1 we find a spot which is in­teresting, we say, "\Ve had better cut out this spot. \Ve had beller make an antibody to it, eventually a monoclonal antibody, alld then sLlrt lookiilg, llsing fluorscencc microscopy, exactly where that antigen is produced and ultimately lISC it as a diagnostic tool if possible."

MAO: One more question: I wonder if you are try;ng to make this new approach oilly for qualitative data or for both qualitative and quantita­tive measurements in your studies?

ANDERSON: At the moment it's qualitative becallse the computer systems arc 110t finished; but ultimately it's quantitati\'c, more or less, for ,111 of the spots.

For instance, during the cell cycle there are not vast qualitative changes in the proteins being synthesized-at least in BELA cells or \VI-38, all you really sec are quan ti ta t i ve c IWllges in the phosphorylat ion of particula r proteins, or in the rate of synthesis of othcr particular proteins. So there are some processes which do not yield gigantic changes. On the other hand, if one starts sticking viruses into cells, or hitting them with Con­canavalin A or something like that, one can get fairly substantial changes in gene expression. That's easier to find by eye!

As a last note, it just occurred to mc to contradict one thing in what was said by the gentleman who was 100 percent correct [Hadden], and it is that this is not exactly futuristic, as he implied. There are a number of peoplc around the world working on systems like this. I tllink once the systems arc fully function~l, it will be qqite easy to arrange to pump a lot of experimental material through them and to collate results fairly quickly. Looking at 500 proteins arlU finding out how each of them varies as a

function of ~Ilything, from the concel1tration of magnesium to erYlhro­poctin, or anylhing else you care to llame) is pretty quick business, once it gets going.