MS1 Peptide Ion Intensity Chromatograms in MS2 (SWATH) Data Independent Acquisitions. Improving Post Acquisition Analysis of Proteomic Experiments

Quantitative analysis of discovery-based proteomic workflows now relies on high-throughput large-scale methods for identification and quantitation of proteins and post-translational modifications. Advancements in label-free quantitative techniques, using either data-dependent or data-independent mass spectrometric acquisitions, have coincided with improved instrumentation featuring greater precision, increased mass accuracy, and faster scan speeds. We recently reported on a new quantitative method called MS1 Filtering (Schilling et al. (2012) Mol. Cell. Proteomics 11, 202–214) for processing data-independent MS1 ion intensity chromatograms from peptide analytes using the Skyline software platform. In contrast, data-independent acquisitions from MS2 scans, or SWATH, can quantify all fragment ion intensities when reference spectra are available. As each SWATH acquisition cycle typically contains an MS1 scan, these two independent label-free quantitative approaches can be acquired in a single experiment. Here, we have expanded the capability of Skyline to extract both MS1 and MS2 ion intensity chromatograms from a single SWATH data-independent acquisition in an Integrated Dual Scan Analysis approach. The performance of both MS1 and MS2 data was examined in simple and complex samples using standard concentration curves. Cases of interferences in MS1 and MS2 ion intensity data were assessed, as were the differentiation and quantitation of phosphopeptide isomers in MS2 scan data. In addition, we demonstrated an approach for optimization of SWATH m/z window sizes to reduce interferences using MS1 scans as a guide. Finally, a correlation analysis was performed on both MS1 and MS2 ion intensity data obtained from SWATH acquisitions on a complex mixture using a linear model that automatically removes signals containing interferences. This work demonstrates the practical advantages of properly acquiring and processing MS1 precursor data in addition to MS2 fragment ion intensity data in a data-independent acquisition (SWATH), and provides an approach to simultaneously obtain independent measurements of relative peptide abundance from a single experiment.Mass spectrometry is the leading technology for large-scale identification and quantitation of proteins and post-translational modifications (PTMs)¹ in biological systems (1, 2). Although several types of experimental designs are employed in such workflows, most large-scale applications use data-dependent acquisitions (DDA) where peptide precursors are first identified in the MS1 scan and one or more peaks are then selected for subsequent fragmentation to generate their corresponding MS2 spectra. In experiments using DDA, one can employ either chemical/metabolic labeling or label-free strategies for relative quantitation of peptides (and proteins) (3, 4). Depending on the type of labeling approach employed, i.e. metabolic labeling with SILAC or postmetabolic labeling with ICAT or isobaric tags such as iTRAQ or TMT, the relative quantitation of these peptides are made using either MS1 or MS2 ion intensity data (4–7). Label-free quantitative techniques have until recently been based entirely on integrated ion intensity measurements of precursors in the MS1 scan, or in the case of spectral counting the number of assigned MS2 spectra (3, 8, 9).Label-free approaches have recently generated more widespread interest (10–12), in part because of their adaptability to a wide range of proteomic workflows, including human samples that are not amenable to most metabolic labeling techniques, or where chemical labeling may be cost prohibitive and/or interfere with subsequent enrichment steps (11, 13). However the use of DDA for label-free quantitation is also susceptible to several limitations including insufficient reproducibility because of under-sampling, digestion efficiency, as well as misidentifications (14, 15). Moreover, low ion abundance may prohibit peptide selection, especially in complex samples (14). These limitations often present challenges in data analysis when making comparisons across samples, or when a peptide is sampled in only one of the study conditions.To address the challenges in obtaining more comprehensive sampling in MS1 space, Purvine et al. first demonstrated the ability to obtain sequence information from peptides fragmented across the entire m/z range using “shotgun or parallel collision-induced dissociation (CID)” on an orthogonal time of flight instrument (16). Shortly thereafter Venable et al. reported on a data independent acquisition methodology to limit the complexity of the MS2 scan by using a segmented approach for the sequential isolation and fragmentation of all peptides in a defined precursor window (e.g. 10 m/z) using an ion trap mass spectrometer (17). However, the proper implementation of this DIA technique suffered from technical limitations of instruments available at that time, including slow acquisition rates and low MS2 resolution that made systematic product ion extraction problematic. To alleviate the challenge of long duty cycles in DIAs, researchers at the Waters Corporation adopted an alternative approach by rapidly switching between low (MS1) and high energy (MS2) scans and then using proprietary software to align peptide precursor and fragment ion information to determine peptide sequences (18, 19). Recent mass spectrometry innovations in efficient high-speed scanning capabilities, together with high-resolution data acquisition of both MS1 and MS2 scans, and multiplexing of scan windows have overcome many of these limitations (10, 20, 21). Moreover, the simultaneous development of novel software solutions for extracting ion intensity chromatograms based on spectral libraries has enabled the use of DIA for large-scale label free quantitation of multiple peptide analytes (21, 22). In addition to targeting specific peptides from a previously generated peptide spectral library, the data can also be reexamined (i.e. post-acquisition) for additional peptides of interest as new reference data emerges. On the SCIEX TripleTOF 5600, a quadrupole orthogonal time-of-flight mass spectrometer, this technique has been optimized and extended to what is called ‘SWATH MS2′ based on a combination of new technical and software improvements (10, 22).In a DIA experiment a MS1 survey scan is carried out across the mass range followed by a SWATH MS2 acquisition series, however the cycle time of the MS1 scan is dramatically shortened compared with DDA type experiments. The Q1 quadrupole is set to transmit a wider window, typically Δ25 m/z, to the collision cell in incremental steps over the full mass range. Therefore the MS/MS spectra produced during a SWATH MS2 acquisition are of much greater complexity as the MS/MS spectra are a composite of all fragment ions produced from peptide analytes with molecular ions within the selected MS1 m/z window. The cycle of data independent MS1 survey scans and SWATH MS2 scans is repeated throughout the entire LC-MS acquisition. Fragment ion information contained in these SWATH MS2 spectra can be used to uniquely identify specific peptides by comparisons to reference spectra or spectral libraries. Moreover, ion intensities of these fragment ions can also be used for quantitation. Although MS2 typically increases selectivity and reduces the chemical noise often observed in MS1 scans, quantifying peptides from SWATH MS2 scans can be problematic because of the presence of interferences in one or more fragment ions or decreased ion intensity of MS2 scans as compared with the MS1 precursor ion abundance.To partially alleviate some of these limitations in SWATH MS2 scan quantitation it is potentially advantageous to exploit MS1 ion intensity data, which is acquired independently as part of each SWATH scan cycle. Recently, our laboratories and others have developed label free quantitation tools for data dependent acquisitions (11, 12, 23) using MS1 ion intensity data. For example, the MS1 Filtering algorithm uses expanded features in the open source software application Skyline (11, 24). Skyline MS1 Filtering processes precursor ion intensity chromatograms of peptide analytes from full scan mass spectral data acquired during data dependent acquisitions by LC MS/MS. New graphical tools were developed within Skyline to enable visual inspection and manual interrogation and integration of extracted ion chromatograms across multiple acquisitions. MS1 Filtering was subsequently shown to have excellent linear response across several orders of magnitude with limits of detection in the low attomole range (11). We, and others, have demonstrated the utility of this method for carrying out large-scale quantitation of peptide analytes across a range of applications (25–28). However, quantifying peptides based on MS1 precursor ion intensities can be compromised by a low signal-to-noise ratio. This is particularly the case when quantifying low abundance peptides in a complex sample where the MS1 ion “background” signal is high, or when chromatograms contain interferences, or partial overlap of multiple target precursor ions.Currently MS1 scans are underutilized or even deemphasized by some vendors during DIA workflows. However, we believe an opportunity exists that would improve data-independent acquisitions (DIA) experiments by including MS1 ion intensity data in the final data processing of LC-MS/MS acquisitions. Therefore, to address this possibility, we have adapted Skyline to efficiently extract and process both precursor and product ion chromatograms for label free quantitation across multiple samples. The graphical tools and features originally developed for SRM and MS1 Filtering experiments have been expanded to process DIA data sets from multiple vendors including SCIEX, Thermo, Waters, Bruker, and Agilent. These expanded features provide a single platform for data mining of targeted proteomics using both the MS1 and MS2 scans that we call Integrated Dual Scan Analysis, or IDSA. As a test of this approach, a series of SWATH MS2 acquisitions of simple and complex mixtures was analyzed on an SCIEX TripleTOF 5600 mass spectrometer. We also investigated the use of MS2 scans for differentiating a case of phosphopeptide isomers that are indistinguishable at the MS1 level. In addition, we investigated whether smaller SWATH m/z windows would provide more reliable quantitative data in these cases by reducing the number of potential interferences. Lastly, we performed a statistical assessment of the accuracy and reproducibility of the estimated (log) fold change of mitochondrial lysates from mouse liver at different concentration levels to better assess the overall value of acquiring MS1 and MS2 data in combination and as independent measurements during DIA experiments.