SPECTROMETRY IN BIOLOGY AND MEDICINE

Appendix

The Meaning and Usage of the Terms Monoisotopic Mass, Average Mass, Mass Resolution, and Mass Accuracy for Measurements of Biomolecules

s. A. Carr, A. L. Burlingame and M. A. Baldwin

Much confusion surrounds the meaning of the terms monoisotopic mass, average mass, resolution and mass accuracy in , and how they interrelate. The following brief primer is intended to clarify these terms and to illustrate the effect they have on the data obtained and its interpretation. We have highlighted the effects of these parameters on measurements of masses spanning the range of 1000 to 25,000 Da because of important differences that are observed. We refer the interested reader to a number ofexcellent articles that have dealt with aspects ofthese issues [1-6].

MONOISOTOPIC MASS VERSUS AVERAGE MASS Most elements have a variety of naturally occurring , each with a unique mass and natural abundance. The monoisotopic mass of an element refers specifically to the lightest stable of that element. For example, there are two principle isotopes of carbon, l2C and l3C, with masses of 12.000000 and 13.003355 and natural abundances of98.9 and 1.1%, respectively; thus the value defined for the monoisotopic mass of carbon is 12.00000 on this mass scale. Similarly, there are two naturally occurring isotopes for nitrogen, l4N, with a mass of 14.003074 (monoisotopic mass) and a relative abundance of99.6% and l5N, with a mass of 15.000109 and a relative abundance of ca. 0.4%. The monoisotopic mass of a molecule is thus obtained by summing the monoisotopic masses (including the decimal component, referred to as the mass defect) of each element present. Of course the measured molecular species (a measurement made on a large, statistical ensemble of molecules) will consist not only of species having just the lightest isotopes ofthe elements present, but also some percent• age of species having one or more of one (or more) ofthe heavier isotopes. The contribution of these heavier isotope peaks in the molecular ion cluster depends on the abundance-weighted sum of each element present. The theoreti• cal appearance of these isotope clusters may be precisely calculated by solving a polynomial expression [6], and there are now many commercially available programs that will do this automatically. On the other hand the chemical average mass of an element is simply the sum ofthe abundance-weighted masses of all of its stable isotopes (e. g., 98.9% l2C and 1.1% l3C, to give the isotope weighted average mass of 12.011 for carbon). The average mass of a molecule is then the sum of the chemical average masses ofthe elements present. The peak top mass is the mass of the peak maximum ofthe isotope cluster. The relationship

553 APPENDIX

Resolullon = 25000 2529.913 Peak Top Mass = 2530.91 Average Mass =2531.67 Monoisotopic Mass

2524 2525 2526 2527 2526 2529 Resolullon = SOOO Peak Top Mass = 2530.91 Average Mass = 2531.67

2524 2525 2526 2527 Resolution = 1000 Peak Top Mass = 2530.93 Average Mass = 2531 .67

2524 2525 2526 2527

Resolution = 500 Peak Top Mass = 2531 .15 Average M... = 2531.67

2524 2525 2526 2527 Resolution = 250 Peak Top Mass = 2531.43 Average Mass = 2531 .67

2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539

Fig. 1. Oxidized b-chain, insulin. Formula C97Hl5lN25046S4' The molecular ion cluster for the oxidized b-chain of insulin is shown at various resolutions. The asymmetry of the cluster becomes less apparent as the resolution is decreased and the peak top mass and the average mass become almost identical. between the monoisotopic mass, average mass, and peak top mass (sometimes referred to as the maximum mass) is illustrated in Figures 1 and 2 for a peptide of mass ca. 2500 and a protein with a mass of ca. 25,000, respectively. One important consequence ofthe contribution ofthe heavier isotope peaks is that for peptides with masses greater than ca. 2000, the peak corresponding to the monoisotopic mass is no longer the most abundant in the isotopic cluster (Fig. 1). With increasing molecular weight, the peak top mass continues to shift upward relative to the monoisotopic mass. Above masses of ca. 8000, the monoisotopic mass has an insignificant contribution to the isotopic envelope (Fig. 2). Whether the monoisotopic mass or the average mass should be used when measuring and reporting molecular weights will depend on the mass of the substance and the resolving power of the mass spectrometer.

RESOLUTION The ability to separate and mass measure signals for peptides, proteins, etc. of similar, but not identical is affected by the resolving power

554 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE

Resolution ~ 5000 Peak Top Mass = 25518.91 Average Mass ~ 25519.58

Resolution = 1000 Peak Top Mass =25579.32 Average Mass = 25519.58

Resolution =25000 Peak Top Mass = 25519.04 Average Mass =25519.58

Resolution = 500 Peak Top Mass ~ 25579.48 Average Mass = 25519.58

2.5545 25550 25555 25560 25565 25570 25575 25580 25585 25590 25595 25600 25605 25610 25615

Fig. 2. Protein HIV-p24. Formula Cl12rJI1802N316033SS13. The molecular ion cluster for the protein HIV-p24 is shown at various resolutions. The position ofthe monoisotopic mass is indicated by the arrow. of the mass analyzer. Because peaks in a have width and shape, it is necessary to evaluate the extent of overlap between adjacent peaks when determin~gthe resolution. Mass resolution is often expressed as the ratio mILlm where m and m + Llm are the masses (in atomic mass units or Da) oftwo adjacent peaks of approximately equal intensity in the mass spectrum (Fig. 3). The height ofthe "valley" between the two adjacent peaks, expressed as a percentage of the height of peak "m", is a measure of the degree of overlap. There are presently two definitions in widespread use, and it is essential to know which is being used when resolution figures are quoted. Historically, the first of these is the so-called 10% valley definition in which the two adjacent peaks each contribute 5% to the valley between them (Fig. 3). In practice, it is often necessary to determine the resolution of the mass analyzer using a single peak (either one peak in a mass• resolved isotopic peak cluster, or one peak corresponding to the envelope of unresolved isotopic peaks, see below). This is accomplished by measuring the peak width (in Da) at the 5% level, and dividing this number into the mass (in Da) of the peak. This definition is most frequently used for molecules with masses below 5000 Da where it is possible on certain analyzers to obtain

555 ApPENDIX

h

8 M @50% h (FWHM)

(10% Valley)

M M+l Fig. 3. resolution of the isotope peaks. For unresolved isotopic peak envelopes the "full• width, half-maximum" (FWHM) definition has come into popular use. The resolution of a peak using this definition is the mass ofthe peak (in Da) divided by the width (in Da) ofthe isotopic envelope measured at the half-height ofthe peak (see Fig. 3). A useful rule of thumb is that the value for the resolution determined using the FWHM definition is approximately twice that obtained using the 10% valley definition (equivalent for singly charged ions to the full• width at 5% peak height for symmetrical peaks). For example, a resolution of 1000 using the 10% valley definition is approximately equivalent to resolution of 2000 using the FWHM definition: clearly, while the resolution value in the latter case is larger, the measured degree of separation of the peaks in Da is identical. It should also be noted that at a resolution of 1000 (FWHM) a peak at mass 1001 will essentially be unresolved from the peak at mass 1000. However, for proteins, resolving the isotopes in the protonated molecu• lar ion envelope is generally not possible (except for FTMS of electrospray• produced molecular ions), nor is it generally useful in practice to be able to resolve the isotopes oflarge molecules like proteins. Furthermore, increasing the resolving power of a mass spectrometer usually reduces the sensitivity of

556 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE

detection. Thus, in sample limited situations, the resolving power of the mass spectrometer may have to be reduced below the level necessary for resolution of the isotopic peaks to allow detection of the protonated molecular ion.

MAss ACCURACY The accuracy of the mass measurement is often stated as a percentage of the measured mass (e. g., molecular mass = 1000 +/- 0.01%), or as parts-per• million (e. g., molecular mass =1000 +/- 100 ppm). As the mass being considered increases, the absolute mass error corresponding to the percent or ppm error will also increase proportionally (e. g., 0.01% or 100 ppm = 0.1 Da at m/z 1000, or 0.5 Da at m/z 5000, or 5 Da at m/z 50,000). Mass accuracy is dramatically affected by how the data is treated by the data system. Very significant error can be introduced in the mass range of 1000 to ca. 5000 if unresolved isotope clusters must be measured. In this mass range, the unresolved clusters will be asymmet• ric in shape, reflecting the asymmetry of the intensity distribution of the underlying isotopic masses in the cluster (see Fig. 1). Ifthe mass being reported is the average mass, one must be sure to measure the centroid of the distribution and not the peak top mass, as these will not generally be the same in this mass range. Errors in measuring the centroid can occur when the ion intensities are weak (due to the inaccurate definition of the peak profile due to poor ion statistics) or due to other distortions of the peak profile of the cluster. Such distortions can be caused by the presence of unresolved adducts with the protonated molecular ion (with, e. g., Na+, K+, photochemical adducts ofMALDI matrices, etc.), or by other proteins of similar but not identical mass that are not resolved. In this case, it will be impossible to obtain an accurate mass assignment unless the instrument resolving power can be increased or the contributing adduct ions can be removed by cleanup of the sample. As the mass of the protein increases, the peak profile (for a pure protein) becomes more symmetrical and the average mass and the peak top mass become almost identical. On the other hand, it becomes more and more difficult to resolve the substance from other substances of very similar molecular masses, and the possibility of adduct formation increases, with a concomitant effect on mass measurement accuracy.

How MUCH RESOLUTION Is REQUIRED? A resolution of at least several thousand (FWHM) is desirable for good mass measurement in the mass range of 1000 to ca. 5000 Da. Why do we care about having sufficient resolution to measure monoisotopic masses? Why not centroid the unresolved peak profile and obtain the chemical average mass? While in theory this should work, in practice it is often difficult with real data to precisely determine the centroid of an unresolved isotope cluster. In order to obtain an accurate centroid it is necessary to define the peak down to the baseline. Unfortunately, this is not routinely achieved as the bottom parts of the clusters are often distorted or poorly defined due to background sample (or matrix in MALDI) components, noise, and poor ion statistics. These factors can easily contribute errors of 1 Da or more in the mass range of 1000 to 5,000 Da.

557 ApPENDIX

In order to use monoisotopic masses you must have adequate resolution to separate adjacent isotope peaks sufficiently that their masses may be individu• ally measured. Resolution sufficient to resolve single mass unit differences is also important for 1) deconvolution of mixtures of very. similar mass (e.g., Asn vs. Asp); 2) being able to distinguish charge-state directly from isotope peak spacing in ESMS (see below), etc. Thus, in the mass range up to ca. 5000 Da it is recommended that as high a resolution as possible be used in order to facilitate mass assignment. If unresolved clusters must be measured, the centroid ofthe isotope cluster should be used to calculate the chemical average mass (while bearing in mind the warnings given above). For example, the resolution obtainable with quadrupole and time-of-flight mass analyzers is insufficient to resolve the individual isotope peaks ofthe elements for molecules with masses in excess of ca. 4000 Da. In this case, an unresolved peak envelope is obtained for a protein, the centroid of which is the abundance-weighted sum (chemical average mass) ofthe isotopes of the elements present. FTMS is unique in being able to provide higher resolution for large molecules (see chapter by Alan Marshall, this volume). Having ultra-high resolution does not help with this problem however, as the monoisotopic peak is of such low intensity that it cannot be identified at these masses. In this higher mass range it is necessary to use the chemical average mass. However, the above statements do not mean that you need only very low resolution at masses above 10,000 Da. The lower the resolution, the lower your ability to detect variants of your protein that differ slightly in mass. Higher resolution also allows the identification of the charge state of an isotope cluster based on the spacing ofthe individual isotopic peaks. Also, in MALDI, one would like to have sufficient resolution to resolve the contribution of any matrix adduct peaks. The relative abundance of such adducts is unpredictable, and they may shift the apparent centroid of the unresolved cluster to higher mass in an unpredictable fashion. The resolution required for species with masses above 10,000 Da is to a first approximate based on the natural width ofthe molecular ion envelope. For a 25 kDa protein the envelope of isotopes is ca. 11 Da wide at FWHM, corresponding to an apparent resolution of2300 (25000/11). Having an instrumental resolution higher than 2300 but less than 30,000 (see Fig. 4) will not improve the apparent resolution because the minimum width of the unre• solved protein molecular ion cluster is determined by the weighted average ofthe natural abundance of the isotopes present. Thus, distinguishing protein variants of similar mass depends on a number of factors in addition to the stated resolution of the mass analyzer, including the absolute mass difference between the components and the width and shape ofthe protein molecular ion "envelopes", which is determined mainly by the mass at which the measurement is being made. The shape and width are also affected by the adduction of small molecular weight ions, such as metal cations or phosphate anions or the photo adduction of matrix molecules often seen in MALDIMS. For example, detection of an N-terminal formyl group (diff. = 28 Da) on a 25 kDa protein would require a resolution ofca. 2000 (FWHM)

558 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE

8- Casein

Average MW = 23982

H'tfH:--r----- 30000 Resolution

,\4\~+---- 2500 Resolution

.....-;.---- 1000 Resolution

..--- 500 Resloution

Fig. 4.

(25,OOO/28 10%valleYdef X 2), which is easily achievable by ESMS on quadrupole mass analyzers. Substantially higher resolution in MALDIMS is typical with a time• of-flight analyzer equipped with an ion mirror or reflectron. Signals due to photochemical adducts of the matrix with the protein are also commonly observed in MALDI. These adducts can give rise to intense signals that, as the MW ofthe protein increases, fold into the (M+H)+ peak of the protein and skew the observed mass to an artificially high value. These adducts also can interfere with detection of variants.

RESOLUTION OF MULTIPLy-CHARGED IONS Electrospray mass spectra of peptides or proteins having several basic sites typically show series of peaks arising from a distribution of multiply-charged ions, each having a different m/z value. For a pure compound the charge states of each ion and the corresponding molecular mass can be determined from the spacing of the peaks [9]. However, some peptides may give only one predominant charge state. Furthermore, the spectrum of a mixture may be too complex to identify ion series that belong to each individual component. In such cases it is possible to determine the charge state of each ion ifthe mass spectrometer has sufficient resolving power , m/~m, to separate the stable isotope

559 ApPENDIX

7 37.93

6 32.28

670 .67

877.93

959.49

600 700 800 90 0 Fig. 5 The partial mass spectrum of an unseparated tryptic digest of 400 fmol of the glycoprotein fetuin. In the inset the peak identified as mlz 877.93 is expanded to reveal a cluster ofisotopic components, separated by 0.25 m I z units, thus the charge state for these ions is 4. profiles. Figure 5 gives an example from the mass spectrum of an unseparated digest of fetuin. This spectrum was recorded at a resolving power of approxi• mately 6,500 FWHM. Expanding the mass scale for the peak identified as rnlz 877.93 reveals a cluster of resolved isotope peaks that differ from each other by 0.25 rnlz units. As these correspond to isotopic peaks with 1 Da mass differences, the charge state can be calculated from the reciprocal ofthe peak spacing, i. e., 4. The first peak in this cluster is at m/z 877.69, thus the monoisotopic molecular mass for the peptide giving this MH44+ion is given by (877.69 x 4) - 4 = 3506.76. This spectrum was recorded on a Qq-TOF mass spectrometer, a hybrid instrument in which the relatively high resolving power (up to 12,000 FWHM) derives from the use ofan orthogonal acceleration TOF analyzer with a reflectron. This mass resolution is sufficient to allow this procedure to be carried out for peptides and small proteins with an upper mass limit of about 8-10 kDa. Qq-TOF instruments are also capable of , in which the precursor ions are selected in the quadrupole analyzer, collided with gas molecules, and the ionic fragments are analyzed in the orthogonal TOF. Selec-

560 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE tion of a multiply-charged precurser ion is likely to give an MSIMS spectrum containing fragments with different charge states. The resolving power of this instrument is the same for fragment ions, thus the charge states can be determined for each peak by inspection of the spacing of each isotope cluster. This information is essential for interpretation of collision-induced dissociation (CID) mass spectra to deduce peptide sequence information.

ACCURATE MAss MEASUREMENTS A term often encountered in the mass spectrometry literature, is the phrase "accurate mass". Historically, this term refers to measurement of ionic masses to an accuracy of 1-10 parts per million (ppm) . This type of accuracy on small molecules allows the assignment of elemental compositions to the masses measured. Recently, it has also been used to refer to the measurement of masses of large molecules, such as proteins, with an accuracy as low as 1 part per thousand. To avoid confusion, it would be better to restrict the use of the term accurate mass to measurements accurate to 1-10 ppm. Further information on this subject is contained in the references listed below [7, 8].

REFERENCES

1. K. Biemann, Meth. Enzymol. 1990,193,295-305.

2. I. Jardine, Meth. Enzymol. 1990, 193,441-455.

3. J. A. Yergey, D. Heller, G. Hansen, R. J. Cotter and C. Fenselau, Anal. Chem. 1983, 55, 353-356.

4. A. Ingendoh, M. Karas, F. Hillenkamp and U. Giessmann, Int. J. Mass Spectrom. Ion Proc. 1994,131,345-354.

5. C. N. McEwen and B. S. Larsen, Rapid Comm. Mass Spectrom. 1992,6, 173-178.

6. J. A. Yergey, Int. J. Mass Spectrom. Ion Proc. 1983,52, 337-349.

7. A .L. Burlingame, R. K. Boyd and S. J. Gaskell, Anal. Chem. 1994,66, 636R.

8. M. L. Gross, J. Am. Soc. Mass Spectrom. 1994,5,57.

9. M. Mann, C. K. MengandJ. B. Fenn,Anal. Chem.1989, 61, 1702-1709.

561 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE

Author Index

Aebersold, R...... 143 Hood, D ...... 439 Albigot, R ...... 463 Hope, J ...... 383 Annan, R ...... 199 Horn, D ...... 111 Arnold, C ...... 365 Huang, L ...... 217 Baillie, T ...... 481 Huddleston, M ...... 199 Bateman, R...... 403 Hunt, D ...... 163 Brando, T ...... 463 Hunter, A ...... 403 Burlingame, A...... 217,289,383 Iyer, S ...... 365 Cantor, C ...... 515 Juhasz, P ...... 1 Carpenter, B ...... 111 Kelleher, N ...... 111 Carr, S ...... 199 Komives, E ...... 91 Carroll, J ...... 365 Kreppel, L...... 365 Cheng, X ...... 365 Kruger, N ...... 111 Chernushevich, 1...... 17 Krutchinsky, A...... 17 Chong, A ...... 383 Kuster, B ...... 403 Christian, R...... 163 Lacomis, L ...... 121 Cole, R ...... 365 Lewis, M ...... 111 Comer, F ...... 365 Li, J ...... 439 Crain, P ...... 531 Loboda, A...... 17 Cramer, R ...... 289 Loo, J ...... 73 Dwek, R ...... 403 Lui, M ...... 121 Emmett, M ...... 31 Mandell, J ...... 91 Ens, W ...... 17 Mann, M ...... 237 Erdjument-Bromage, H ...... 121 Marshall, A...... 31 Falick, A ...... 91 Martin, A...... 439 Figeys, D ...... 143 Martin, S ...... 1 Freckleton, G ...... 121 Martin, S E...... 163 Freitas, M ...... 31 Marto, J ...... 163 Fridriksson, E...... 111 McCloskey, J ...... 531 Geromanos, S ...... 121 McLafferty, F ...... 111 Gilleron, M ...... 463 McNulty, D ...... 199 Goodlett, D ...... 143 Mei, H ...... 73 Grewal, A...... 121 Monsarrat, B...... 463 Gygi, S ...... 143 Moseley, M ...... 179 Hart, G ...... 365 Moxon, E ...... 439 Harvey, D ...... 403 Muller, E ...... 271 Haynes, P ...... 143 Murphy, R...... 497 Hendrickson, C ...... 31 Nakamura, T ...... 497 Herring, C ...... 309 Nettleton, E ...... 53 Hines, W ...... 1 Nigou, J ...... 463 Ho, Y ...... 531 Northrop, D ...... 329

563 AUTHOR INDEX

Otto, A ...... 271 Parker, G ...... 365 Pearson, P ...... 481 Philip, J ...... 121 Posewitz, M ...... 121 Puzo, G ...... 463 Qin,J...... 309 Qiu, F ...... 531 Richards, J ...... 439 Robinson, C ...... 53 Rozenski, J ...... 531 Ruffner, D ...... 531 Russo, P ...... 163 Settlage, R ...... 163 Shabanowitz, J ...... 163 Shevchenko, A...... 237 Simpson, F ...... 329 Standing, K ...... 17 Stimson, E ...... 383 Tempst, P ...... 121 Thanabal, V ...... 73 Thibault, P ...... 439 Vestal, M ...... 1 Wang, C ...... 217 Watts, J ...... 143 Wheeler, S ...... 403 White, F ...... 163 Wittmann-Liebold, B...... 271 Zhang, X ...... 309 Zhang, Z ...... 31 Zubarev, R ...... 111

564 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE

Subject Index

A accelerator mass spectrometry (AMS) ...... 492 accuracy, mass measurement ...... 40 adducts ...... 31 adsorptive loss ...... 125 affinity chromatography, immobilized metal ion ...... 127 gas phase ...... 89 purification ...... 241 amyloidosis, senile systemic ...... 55 amyloid plaques ...... 53 angiotensin II peptide ...... 152 anion-binding-exosite I ...... 101 antigen 164 apoptosis Burkitt lymphoma cell ...... 271 protein marker ...... 278 arachidonic acid ...... 497 epoxyeicosatrienoic acid ...... 502 automatable ...... 74

B

~-elimination ...... 153 beta lactoglobulin ...... 169 binding interactions hydrophobic ...... 83 electrostatic ...... 83 bioinformatics ...... 121, 180 bovine serum albumin ...... 169, 191 bradykinin ...... 42 Burkitt lymphoma ...... 271, 273 c

C22A-FKBP ...... 35 calcium ...... 80, 81 calculation, percent inhibitor bound ...... 109 capillary electrophoresis (CE) ...... 464 capillary column ...... 182

565 SUBJECT INDEX

carbohydrate ...... 403 acidic ...... 410 alkali metal ...... 407 derivatization ...... 414 high energy ClD ...... 428 MALDl ...... 403 MALDl matrix ...... 407 MSIMS ...... 424 post-source decay ...... 435 quantitative analysis ...... 420 carbonic anhydrase ...... 149, 169 casein ...... 169 CD40 ...... 170 cdc42 protein ...... 43 cDNA sequence error ...... 230,231 cell cycle control ...... 257 state of ...... 144 charge state ...... 60 chemical compound library ...... 82 chemical reaction interface mass spectrometry (CRlMS) ...... 492 ClD (see collision-induced dissociation) citrate synthase ...... 20 clean-up ...... 142 cleavage, disulfide bond ...... 115 collisional damping ...... 19 collision-induced dissociation (ClD) high energy ...... 2 spectra, high energy ...... 7 complex HlV Tat peptide-TAR RNA ...... 81 insulin hexamer ...... 88 mixtures ...... 74 non-covalent ...... 31, 76 water molecules ...... 62 quaternary protein ...... 83 streptavidin tetramer ...... 77 Tat-Tar ...... 81,82 concentration ligand ...... 109 protein ...... 109 sample ...... 142 cone voltages ...... 58 conformational dynamics ...... 55 contamination, keratin ...... 176

566 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE control posttranslational ...... 144 translational ...... 144 corona discharges ...... 75 covalent modification dynamic ...... 144 posttranslational ...... 144 reversible ...... 144 cross section, electron capture ...... 115 cytochrome C ...... 169

D dalargin ...... 25 database search ...... 245, 276 peptide map ...... 247 deamidation ...... 31 delayed extraction ...... 13 denaturant ...... 145 de novo sequencing ...... 226 derivatization acetolysis ...... 469 methylation ...... 253 reductive amination ...... 464 desalting ...... 245, 414 detector, PATRIC ...... 75 deuteration, amide ...... 104 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid ...... 76 digestion, protein ...... 146 dissociation blackbody infrared ...... 112 constant ...... 348 electron capture (ECD) ...... 111 infrared multiphoton ...... 111 non-ergodic ...... 117 distortions, space charge ...... 39 disulfide bridge ...... 31 DNA array ...... 121 high throughput ...... 517 MALDI ...... 517 duplex ...... 83 sequencing ...... 527

567 SUBJECT INDEX drug discovery ...... 74 -drug interaction ...... 485 metabolism ...... 481 cassette dosing ...... 485

E electron, low energy ...... 112, 113 (ESI) ...... 111, 329, 334, 383 CE ...... 443,465 nano ...... 243,249 LC ...... 385,546 nozzle voltage ...... 538 orifice potential ...... 443 electrostatic forces ...... 75 enzyme catalase HPII ...... 20 enzyme kinetics ...... 329 inhibition ...... 348 rapid mixing ...... 343 ESI (see electrospray ionization) EST analysis ...... 121 excess energy, electron capture ...... 115 exoglycosidase digestion ...... 385, 392 extracellular matrix ...... 78 extraction polypeptide ...... 123 RP micro-tip ...... 124

F

FK506-binding protein ...... 34 flow rate, low (-20 nUmin) ...... 135 footprinting experiments ...... 82 formic acid ...... 198 Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) ...... 31 dynamic range ...... 36 signal to noise ...... 36 free energy ...... 347 free radical reaction ...... 497 FT-ICR-MS, FTMS (see Fourier transform ion cyclotron resonance mass spectrometry)

568 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE

function ...... 179 functional assay ...... 141 diversity ...... 232

G

Ga(III) affinity ...... 142 ions ...... 129 galardin ...... 80 gas chromatography-mass spectrometry (GC-MS) ...... 464 gel electrophoresis ...... 145 2-D ...... 219, 241, 275 gel stain Coomassie ...... 174, 219 silver ...... 148, 174, 219 gene knock-out ...... 141, 179 tagging ...... 239 genome project ...... 515 polymorphism ...... 520 transcript profiling ...... 526 genomics ...... 140 glu-l-fibrinogen peptide ...... 4, 184 glycan ...... 366 glyceraldehyde phosphate dehydrogenase ...... 169 glycolipid ...... 439 glycopeptide ...... 296, 386, 397 exoglycosidase digestion ...... 385, 392 glycoprotein ...... 365,383 ~-elimination ...... 375 glycan profiling ...... 417 glycosidase ...... 411 hydrazinolysis ...... 411 MALDI sensitivity ...... 374 N -linked ...... 403 O-GlcNAc ...... 365, 370 glycosylation ...... 31, 365 GPI-anchor ...... 368 GRASP program ...... 95 green fluorescent protein (GFP) ...... 243

569 SUBJECT INDEX

H

Haemophilus in{luenzae ...... 439 strain ...... 450 IIID exchange ...... 31, 96, 98, 107 gas phase ...... 36 off-exchange ...... 103, 107 rate constants ...... 36 high throughput analyses ...... 121, 180 HIV-1 protease ...... 76,79 human a-thrombin ...... 92, 100, 106 thrombomodulin [TM(4-5)] ...... 92, 100 hydrazinolysis ...... 443 hydrogen radical, excited ...... 117 hydrophobic interactions ...... 75 hydroxamate ...... 80

I immonium ion ...... 8 immunochemistry ...... 141 immunoproteasome ...... 218 information management ...... 136 in-gel digestion ...... 242, 243, 245, 299, 311 choice of enzyme ...... 323 glycoprotein ...... 412 inhibitors, phosphopeptide ...... 89 insulin hexamer complex ...... 88 interactions ...... 87 ligand-target ...... 84, 104 protein-protein ...... 92 ion charge ...... 33 desolvation ...... 76 selection, primary resolution ...... 2 trap ...... 313 types c ...... 111 z· ...... 111 isoelectric focusing ...... 145 isoform ...... 232 isotope, 180 ...... 256 isotopic envelope ...... 105

570 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE

J,K

KD-values ...... 109 keratin ...... 176, 249

L labile macromolecule ...... 289 lipid peroxidation ...... 507 lipoglycan ...... 463 MALDI ...... 467 ManLAM ...... 465 PIM2 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 470 lipopolysaccharide ...... 439 extraction ...... 442 hydrazinoloysis ...... 443 O-deacylation ...... 443 liquid chromatography-mass spectrometry (LC-MS) 1,1,1,3,3,3-hexafluoro-2-propanol ...... 535 liquid chromatography-nuclear magnetic resonance-mass spectrometry (LC- NMR-MS) ...... 492 low copy number ...... 141 lysozyme ...... 53,54 M

MALDI (see matrix-assisted laser desorption ionization) mass spectrometer double focusing hybrid ...... 75 Fourier transform ...... 166 LCQ ion trap ...... 165 Manitoba ESI-TOF ...... 18 Q-TOF ...... 184, 256, 275, 387 Sciex API-III triple quadrupole ...... 184 matrilysin ...... 80 matrix-assisted laser desorption ionization (MALDI) ...... 93, 96, 243 carbohydrate ...... 408 DHB ...... 312, 408 in-source decay ...... 119 IR ...... 289 glycopeptide ...... 296 matrix ...... 290 phosphopeptide ...... 299 post-source decay ...... 307 resolution ...... 291

571 SUBJECT INDEX melittin ...... 25 metalloproteinases ...... 78 MHC molecules ...... 164 micro-biochemistry ...... 121 micro-column ...... 142,229 microdroplets ...... 60 microfabricated device ...... 147 molecular interactions ...... 74 monohydroxyeicosatetraenoic acid ...... 500 monoisotopic ...... 31 species ...... 39 mRNA 179 level ...... 162 MS-MS carbohydrate ...... 424 CE ...... 146 CE-ESI ...... 443 ESI, negative ion ...... 500 LC ...... 146,190 ESI ...... 313 fatty acid ...... 509 quantitative ...... 484 nanD ESI ...... 249, 253, 279, 397 tandem TOF ...... 2 MS-Tag ...... 11,220 mucin 367 multiprotein complex ...... 237 cyclosome ...... 259 murine cAMP-dependent protein kinase ...... 92 mutagenesis, site directed ...... 101 Mycobacterium tuberculosis ...... 463 myoglobin ...... 26 myoglobin-heme ...... 83

N

N-acetyl-glucosamine ...... 153 nanospray interface, coaxial ...... 180 neomycin B ...... 81, 82 neuropeptide ...... 215 nitrocellulose ...... 245 nomenclature, 20S proteasome subunits ...... 219 noncovalent binding ...... 76 interactions ...... 17

572 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE northern blots ...... 141 N-terminal nucleophile, Thr-l ...... 218 o

O-GlcNAc modified peptide ...... 154 oligonucleotide ...... 531 2'-O-methyl modification ...... 545 nearest neighbor determination ...... 543 sequencing ...... 539 oligosaccharide ...... 365, 403, 439 orexin-A ...... 201, 203 orthogonal injection ...... 18 p parent ion scanning ...... 443 leucine/isoleucine ...... 249 Paul trap ...... 119 ...... 38,119,177 pepsin ...... 34, 94 pepstatin A ...... 76 inhibitor ...... 79 peptide ACTH(I-17) ...... 6 map ...... 247 sequence, de novo ...... 199 sequence, Edman ...... 199 perfusion beads, metal ion-derivatized ...... 122 phenotype ...... 179, 199 phosphatase PPI ...... 170 PP2A ...... 170 treatment ...... 312 phospholipid ...... 497 phosphopeptide ...... 126, 299 98-Da loss ...... 313 LC MSIMS ...... 313 MALDI ...... 316 MSIMS ...... 313 sensitivity ...... 324 Siclp ...... 208 TOF stability ...... 316 phosphoprotein ...... 151

573 SUBJECT INDEX phosphorylation ...... 31, 121, 309 post-column eluant pH modification ...... 195 surface tension ...... 195 posttranslational modification ...... 121,201,215 peptide ...... 293 posttranslational processing ...... 219 precursor ion scan analysis, rn/z 79 ...... 210 Pro12-Ala-Arg ...... 9 prostaglandin ...... 498 protease inhibitor ...... •... 490 pepsin ...... 34,94 proteasome 208 complex, rat liver ...... 217 subunit composition ...... 218 protein ...... 75 actin ...... 281 APP ...... 372 ~-lactamase ...... 335 caspase ...... 272 Cdc20p ...... 239 complex, dissociation ...... 77 complex, functional ...... 150 concentration ...... 109 digestion ...... 146 doubly depleted, 13C,15N, ...... 39 EP8P synthase ...... 354 expression level ...... 162 fetuin ...... 295 folding ...... 104 function ...... 201 glycosylation ...... 365 hexokinase ...... 337 isolation, native source ...... 144 isotopic depletion ...... 34 kinase A (PKA) ...... 105 lactase ...... 350 low abundance ...... 149 lysozyme ...... 351 machine, proteasome ...... 217 measured molecular weight ...... 230 membrane ...... 89 MIHCK ...... 314 mixture ...... 247

574 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE protein (cont.) modified proteolytic fragment ...... 122 p16 tumor suppressor ...... 33 phosphorylation ...... 309,372 proteolytic fragment ...... 122 PrP ...... 383 ribosomal ...... 248 Rrp4p ...... 249 Rrp43p ...... 249 solubility ...... 145 solvent accessible surface ...... 95 Ssa2p ...... 246 STAT5A ...... 325 synapsin ...... 373 tau ...... 372 TIMP-l ...... 419 xylanase ...... 355 proteoglycan ...... 367 proteome project carcinoma ...... 143 D. discoideum ...... 143 E. coli ...... 143 fibroblast ...... 143 H. in{luenzae ...... 143 keratinocyte ...... 143 kidney ...... 143 liver ...... 143 M. tuberculosis ...... 143 O. anthropi ...... 143 plasma ...... 143 rat serum ...... 143 R. leguminosarum ...... 143 S. cerevisiae ...... 143 S. enterica ...... 143 S. milliferum ...... 143 Synechocystis spp ...... 143 proteomics ...... 140, 143, 163, 179 proton exchange, amide ...... 91 neutralization ...... 115 PSD spectrum ...... 226

Q

Qq-TOF ...... 226

575 SUBJECT INDEX

quadrupolar axialization ...... 42 quaternary protein complexes ...... 83 quinazoline ...... 82

R

recovery, peptide ...... 123 renin substrate ...... 25 resolving power, ultrahigh mass ...... 34 RNA ...... 75 polymerase, alpha subunit ...... 32 posttranscriptional modification ...... 531 ribozyme cleavage ...... 533 s

SAGE ...... 121 sample concentration ...... 142 introduction, automation ...... 195 sequence database ...... 146 SEQUEST ...... 167 sheath liquid ...... 197 Sic1p ...... 201 software, data dependent ...... 174 solubility, protein ...... 145 solvated ions ...... 77 solvent accessibility ...... 104 S-phase Cdk inhibitor ...... 201 src SH2 ...... 89 stoichiometry ...... 74,80,82,88 stromelysin ...... 78, 80 substance P ...... 25 sulfur hexafluoride ...... 75 surface area, solvent accessible ...... 104 SWIFT ...... 38 T

Tat peptide ...... 76 TFA ...... 198 31-mer, TAR RNA ...... 76, 81 thrombin peptide ...... 105 thyroxine ...... :...... 56,62

576 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE

timed-ion-selector ...... 4 time-of-flight (TOF) tandem ...... 2 TOF (see time-of-flight) topology ...... 31 transthyretin ...... 53 trypsin ...... 165 TTR tetramer ...... 55 tumor ...... 121 tuning conditions, ESI interface ...... 77 two-hybrid system ...... 121 u

ubiquitin ...... 32,44, 111 ultracentrifugation ...... 66 v

variants, amyloidogenic ...... 65 w

water molecule ...... 78, 88 y

yeast G6PD ...... 123 z

zinc ...... 80, 81 binding sites ...... 80 Z-Spray source ...... 185

577