1702
Anal. Chem. 1989, 61, 1702-1708
Interpreting Mass Spectra of Multiply Charged Ions Matthias Mann, Chin Kai Meng, and J o h n B. Fenn*
Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520-2159
We descrlbe two algorHhms that extract molecular mass Informatlon from spectra showlng sequences of peaks due to ions wRh varylng numbers of charges. The flrst, called here the “averaglng atgortthm”, unambiguously asslgns charge numbers to the Ions assoclated with the m / z value for each peak In the sequence and then averages the resuttlng values of M to glve a best eotlmate d the molecular mass. The second, ldentlfled as the “deconvolutlon algorlthm”, mathematically transforms a spectrum of several peaks for muttlply charged lono Into one peak correspondkrg to a slngiy charged ion. The procedures can be readily Implemented wRh a personal computer and are here applied to representatlve spectra of small proteins generated by electrospray mass spectrometry. These algorl#rms are now routinely used In our laboratory for the lnterpretatlon of such spectra. They both are fast and convenlent, discriminate agalnst beckground, and take advantage of much of the lnformatlon contalned In a sequence of peaks. Achievable accuracy and sources of error are discussed.
I. I N T R O D U C T I O N The ions produced by the sources traditionally used in mass spectrometry generally comprise singly charged species resulting from the loss or gain of an electron by a parent molecule. Moreover, an appreciable fraction of the ions are often charged fragments of the parent molecule. On the other hand, ions produced by some of the more recently developed sources consist of neutral parent molecules to which small cations or anions are attached. Among these newer and “softer” ionization methods are electrohydrodynamic ionization (EH), fast atom bombardment (FAB), fast ion bombardment (FIB) commonly referred to as secondary ion mass spectrometry (SIMS), laser desorption (LD), plasma desorption (PD), thermospray (TS), and aerospray (AS) originally known as atmospheric pressure ion evaporation (APIE). Due in part to the larger size of the molecules that can be accommodated by these new sources and in part to the nature of their ionization processes, ions containing up to five or six adduct charges have been observed ( 1 ) . However, to our knowledge, except for some preliminary work in our laboratory, no study on how to make efficient use of the peak multiplicity has been reported (2). Recently, with an electrospray (ES) mass spectrometer that has been previously described (3),we have been able to obtain the mass spectra shown in Figure 1 for eight small proteins with molecular weights from 5000 to almost 40000 ( 4 , 5). Analyte samples were dissolved in solvents comprising mixtures of acetonitrile, water, and methanol or 1-propanol. It was necessary to lower the solution pH by addition of small quantities of acetic acid (HAC)or trifluoroacetic acid (TFA). The optimum proportions of these solvent components depended somewhat on the particular sample and were determined by trial and error. Solutions with analyte concentrations ranging from 0.7 to 137 pmol/L, depending upon the species, were injected at flow rates of 8 pL/min. Each of the spectra shown is the result of a single scan requiring 30 s to cover the indicated mass range. The analyzer was a VG
Micromass 1212 with a nominal upper limit for m / z for 1500. The analog output from the Channeltron detector was digitized with an analog to digital converter and fed into a homemade data recording and processing system based on an IBM-AT clone. Since our preliminary report at the ASMS Meeting in San Francisco last June, two other groups have confirmed our results (6,7). Indeed, Edmonds et al. were able to obtain ES spectra for a bovine albumin dimer with a molecular weight of 133000. Although the experiment was not optimized for sensitivity, it is apparent from Table I and Figure 1 that very low detection limits can be achieved. For example, the spectrum of lysozyme consumed only about 3 pmol of sample although more was used because processing and manipulation were not very efficient. In each case the spectrum comprises a sequence of peaks with an intensity distribution that is near Gaussian, has a width of around 500 on the m / z scale, and is generally centered at a value between 800 and 1200. The constituent ions of each peak differ from those of its adjacent neighbors by one elementary charge. For the reader’s convenience we have shown the number of such charges per ion for two or three peaks in each spectrum. Each such charge is due to an adduct cation from the original solution. Our analyzer did not have sufficient resolution for large ions at these m / z values to permit an unequivocal assertion of unit mass for an adduct ion. However, the need for low pH in the sample solution, along with results obtained for smaller peptides and amino acids, strongly support our assumption that H+is the most likely charge carrier in these experiments. For the eight proteins we studied Table I summarizes the essential features of each spectrum and the information it provides. It is immediately apparent from the figures and the table that the degree of multiple charging in ES ionization is much higher than has been encountered with any other soft ionization method. This feature is very attractive in that it extends the effective mass range of any analyzer by a factor equal to the number of charges per ion. -Moreover,because the ions have lower m / z values, they are generally easier to detect and weigh than are singly charged ions of the same mass. On the other hand, peak multiplicity distributes the signal for one species over several channels. But because the number of charges per ion is almost always greater than the number of peaks, the total current carried by one species is greater when there is peak multiplicity than would be the case for a single peak containing the same total number of singly charged ions. Unfortunately, we do not yet know the detector response per charge of a multiply charged ion. We do know, however, that no postacceleration has been required for multiply charged ions that were large enough to require such acceleration had they been singly charged. We also know that the detection sensitivity obtained with ES ionization of large molecules seems to be substantially greater than has been obtained with sources giving rise to ions that are predominantly singly charged (8). Moreover, as will emerge in the subsequent discussion, because peak multiplicity allows signal averaging, mass assignment can be made with more precision and confidence than would be the case for a single peak of a singly charged ion. The objective of this paper is to present basic methods for interpreting the sequence of multiply charged peaks and to provide algorithms for retrieving the
0003-2700/89/0361-1702$01.50/00 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 15, AUGUST 1, 1989
1703
Table I. Data for the Spectra in Figure 1 concentration g/L Fmol/L
mol w t insulin (bovine) cytochrome c (horse heart) lysozyme (chicken egg) myoglobin (equine skeletal muscle) trypsin inhibitor (soybean) a-chymotrypsinogenA (bovine pancreas) carbonic anhydrase I1 (human erythrocytes) alcohol dehydrogenase (horse liver)
5 733 12 360 14 306 16 950 20 091 25 656 29 006 39 830
0.05 1.67 0.01 1.00
0.10 0.50 0.50 0.50
8.8 137 0.71 58.8 5.0 19.0 17.2 12.5
charges
m / z range
4-6 12-20 10-15 15-27 16-22 17-22 23-36 32-46
950-1450 600-1100 900->1500 600-1400 800-1400 1150-> 1500 725-1500 800-1300
The molecular weight was determined from the sequence information provided mostly by ref 8 and is an average value based on the natural abundance of isotopes.
400
0
'11
Lysozyme
1600
1200
800
900
700
1100
1300
IO
= 14,306
M.W.
500
8
6
or
6
4
4-
-
15"
20
1
0
lo
1
,
1
2 1
800
400
,
0
1
1200
1600
Typrln lnhlbltor M.W. = 20.091
8
400
11
8
1
M.W. = 29,006
6
6
4
4
2
2
0 700
0 700
1100
a-Chyrnolyprlnogen A M.W.
=
25,656
1000
1200
1400
1600
where Ki is the apparent value of m / z for the peak position on the scale of the mass analyzer and K
