Analysis of the stochastic variation in LTQ single scan mass spectra

High throughput proteomics requires high speed data acquisition. Signal-averaged spectra have relatively cleaner signals and therefore produce more reliable sequencing results than single scan spectra. However, collecting signal-averaged spectra is also more time-consuming, which may not be feasible for many proteomics applications that are duty cycle limited, i.e. limited by the scanning speed of the instrument and the need to collect spectra for as many peptide precursor ions as possible. The single scan spectra can be viewed statistically as components of the signal averages, i.e. the signal average with random error. In a properly functioning instrument the scan-to-scan variation on the m/z scale is small. For our LTQ it is within the manufacturer’s specification of 0.1 to 0.2 m/z units, but the peak intensities are highly variable. Due to a lack of a proper way to deal computationally with the large scan-to-scan variation in signal intensity, many currently available database matching programs, including SEQUEST1, depend primarily on the m/z axis (is the ion present above a certain threshold or not?) to match sequences and do not make extensive use of relative abundance differences among ions within the same spectrum. However, the intensities carry additional information beyond m/z value2, and incorporating this dimension of information can enhance the quality of the spectral match to a theoretical sequence calculated from a protein database. For instance, Zhang3 incorporated the similarity of intensities between the predicted spectra and the observed spectra in his reported scoring function. However, Zhang and others have normally used signal-averaged data to develop new computational approaches to using tandem mass spectrometry in conjunction with protein databases. Recent improvements in mass spectrometer technology have increased scan speed and the quality of single scan spectra to the point that their use has become practical for high throughput applications, something that was normally not done with the previous generation of commercial quadrupole ion trap mass spectrometers. Studying the noise variation in such single scan spectra can help gain a better understanding of the relationship between signal and noise in the new instruments, can facilitate extraction of the peptide sequence information the intensity carries, and can eventually improve performance of peptide sequencing/database searching software that uses single scan CID mass spectra as an input. The noise characteristics and other fundamental aspects of detectors in mass spectrometry have been extensively studied for many years. The literature up to 1979 was summarized in the doctoral dissertation by Peterson4. Examples of published reviews include those by Geno5 and Evans6. Linear ion trap instrumentation, with an emphasis on mass analyzer design, has recently been reviewed by Douglas, Frank and Mao7. An early version of the LTQ, similar to the instrument used in our laboratory, but with a single detector geometry and less advanced electronics, has been described by Schwartz, Senko and Syka8. The dual detector design employed in the LTQ has been described in the manufacturer’s documentation9. For a properly functioning instrument of the type used here, the variation on the m/z scale is usually a small quantity (< 0.2 m/z units), and to a first approximation is independent of the m/z value under our conditions. For mass spectrometers used with conventional detectors, the intensity of a peak at a certain m/z is directly proportional to the count of ions detected within a predetermined time unit, the length of time associated with the particular scan event. Theoretically, such a detection process often follows a Poisson distribution, in which the standard deviation of intensity scales linearly with the square root of the intensity4, 5, 6. More specifically, the theoretical distribution of ions forming a peak in the mass spectrum can be defined by a Poisson random variable that is determined by the physical and stochastic properties of the specific detector. In this study, we provide a systematic assessment of the noise properties of single scan spectra from the LTQ, from multiple product ion scans for five different precursor ions of different lengths, amino acid compositions and charge distributions. We believe these spectra to be adequately representative of the vast majority of peptides in our archives for purposes of studying scan-to-scan intensity variations. To correct for the slight scan-to-scan variation on the m/z axis, we applied a matching algorithm to align the ions from different scans prior to the analyses of signal intensity variation. The data were normalized so that the total intensity of all ions summed is equal to 1 on a linear scale. This normalization is more stable than the standard method of scaling the intensities so that the highest peak has intensity 100%. The aligned spectra were then used to study both variation on the m/z scale and the variation in intensity. Section 2 describes the data collection methods, the matching algorithm and other statistical methods. Section 3 presents the results and Section 4 includes discussion.