Use of Linear Ion Traps in Data-Independent Acquisition Methods Bene ﬁ ts Low-Input Proteomics

: The need for a better understanding of cellular heterogeneity has pushed mass spectrometry technologies to the analysis of single-cell and single-cell-type proteomes, although several challenges still limit their widespread implementation. Among the e ﬀ orts toward single-cell and low-input analyses, there is the adoption of data-independent acquisition methods to increase analytical sensitivity. Here, we revisited the use of linear ion traps mass analyzers in data-independent acquisition methods and demonstrate their bene ﬁ ts to boost peptide and protein identi ﬁ cations in low-input proteomes.

S ingle-cell and low-input applications have recently exploded as the means to better understand the cellular heterogeneity that drives the development of normal and pathological tissues. 1 Although a plethora of methods have flourished in genomics, 2 single-cell and low-input technologies still face several challenges in mass spectrometry-based proteomics, both at sample handling and data acquisition.A few efforts have successfully been made in both directions 3−7 that have paved the road for these proteomics technologies, but there are still several challenges that limit their widespread implementation. 8mong the efforts toward the analysis of single-cell and single-cell-type proteomes, there is the adoption of dataindependent acquisition (DIA) methodsoften combined with ion mobility separationto increase the sensitivity during the analysis of small amounts of complex samples. 3,6DIA methods use several broadband isolation windows to co-isolate and fragment peptides within samples regardless of their signal or amount.Consequently, these methods provide a systematic sampling of fragment ions that are later used for peptide identification and quantification with high sensitivity and improved dynamic range. 9−11 Despite several DIA methods were developed using linear ion traps in their origins, 12,13 most DIA methods currently rely on high accuracy/high resolution mass analyzers such as time-of-flight and Orbitrap, 14−18 with few exceptions. 19Linear ion traps offer fast scanning rates and high sensitivity that exceed those of high accuracy/high resolution orbital ion traps.Therefore, here we revisited the use of linear ion traps mass analyzers in DIA methods to evaluate their potential to boost peptide and protein identifications in low-input proteomics applications.
Initially, we made serial dilutions of commercially available HeLa protein digest and loaded 1, 10, 100, and 1000 ng in a 50 cm nano-LC column for liquid chromatography separation and subsequent mass spectrometric analysis with an EASY-nLC 1200 coupled to an Orbitrap Fusion Lumos (Supporting Information).A gradient of 120 min was used to resolve the proteomes and obtain peak widths compatible with the cycle times of the evaluated methods (Table 1).All dataindependent acquisition methods tested in this study had their precursor peptides ions analyzed in a high accuracy/high resolution Orbitrap mass analyzer, whereas fragment ions generated by HCD fragmentation (NCE 28) were analyzed either in an Orbitrap or a linear ion trap mass analyzer.−22 An Orbitrap-based DIA method with 40 windows of 10 Da covering the range of 500−900 m/z at 30,000 resolution (DIA-OT 40X10 30K) was used as the reference method and compared to equivalent methods using the linear ion trap for the analysis of the peptide fragment ions (DIA-IT 40X10).Several linear ion trap-based DIA methods (DIA-IT) at different scanning speeds were tested, i.e., normal, rapid, and turbo (Table 1).In all methods the injection times were adjusted to match the scanning speeds and thus achieve optimal ion management.Acquired mass spectrometric data were analyzed with Spectronaut v14.7 (Biognosys) using the directDIA approach and the Pulsar search engine, 22 with peptides and proteins filtered at 1% FDR.Our results showed that linear ion trap DIA methods outcompeted Orbitrap-based DIA (DIA-OT) methods on the number of precursors identified when analyzing low amounts of sample (<10 ng) (Figure 1A).Moreover, within the DIA-IT methods, we observed that those using slightly slower scanning rates and larger MS2 injection times resulted in a higher number of peptide identifications.These observations were further corroborated with additional triplicate analyses with 1 ng of HeLa tryptic digest (Figure 1B).These analyses confirmed that the higher sensitivity exhibited by linear ion traps in DIA methods are beneficial in terms of peptide identification in low-input proteomes.Beyond the increase in the number of peptide identifications, we further evaluated whether DIA-IT methods recapitulated the same results than DIA-OT methods in terms of identified peptides and their quantification.Our results showed an excellent overlap between DIA-IT and DIA-OT methods in the set of identified peptide sequences (Figure 1C).To evaluate the correctness of the peptide identifications, we corroborated that the precursors identified in low-input samples with the DIA-IT method correspond to the most abundant peptides within the sample (Supplementary Figure 1A), and we confirmed that their retention time values highly correlate among the different assessed methods (Supplementary Figure 1B).Moreover, we inspected the pseudo-MS2 spectra created from the directDIA algorithm.Not only are the spectra obtained in low-input samples with DIA-IT methods comparable to those obtained with DIA-OT (Supplementary Figure 2), but the ion-trap spectra have in general more annotated fragment ions than those spectra resulting from Orbitrap-based DIA methods when analyzing low-input samples (Supplementary Figure 3).In terms of peptide quantification, our results showed similar or even better distribution of coefficient of variation in DIA-IT triplicate analyses (Figure 1D) and a very good correlation in terms of peptide quantification (Figure 1E).Finally, it is worth noting that even the slowest evaluated DIA-IT method (DIA-IT 40X10 normal) exhibited reduced cycle times (1.1 s in turbo, 1.3 s in rapid, and 1.9 s in normal) compared to the reference DIA-OT method (3.5 s) and increased number of average sampling points per chromatographic peak (Figure 1F, Supplementary Figure 4A).
We reasoned that if the improvement in peptide identification observed in low-input proteomes was mainly due to higher sampling rates, a similar effect could be achieved by reducing the mass resolution in the DIA-OT methods and thus obtain enough data points in low-intensity chromatographic peaks.We therefore tested variants of the DIA-OT method with reduced resolution (15K and 7.5K) and adjusted injection times for optimal ion management.These Orbitrapbased DIA variants exhibited shorter cycle times (2.2 s in 15K, 1.3 s in 7.5K) and higher sampling rates than the reference DIA-OT method.However, the use of low-resolution, fastscanning DIA-OT methods proved to be less sensitive in all the amounts of the measured HeLa tryptic digest (1−1000 ng) (Supplementary Figure 4B).Indeed, these methods performed worse in terms of peptide identifications than the linear ion trap-based methods, and any gain achieved from faster scanning speeds could not compensate the combination of low resolution, smaller companion injection times and the lower sensitivity of the Orbitrap mass analyzer.
Next, we wanted to confirm that the observed benefits of linear ion trap-based DIA methods were independent of the isolation scheme used.For this purpose, we adapted the recently published DIA+ method 23 by setting the linear ion trap as the mass analyzer for peptide fragments and matched the MS2 injection times for optimal method synchronization (Supporting Information).DIA+ is a data-independent acquisition method with nonsequential multiplexed isolation windows that combine signals from identical peptides with different charge states, and therefore, it differs considerably from the isolation scheme previously evaluated.We performed triplicate analyses of 1 ng of HeLa tryptic digest with the reference Orbitrap-based DIA+ method and compared it to the equivalent methods using the linear ion trap for the analysis of the peptide fragment ions.Our results showed that the linear ion trap-based DIA+ methods also outperformed the reference Orbitrap-based DIA+ method in terms of peptide identifications.Despite the intrinsic lose of specificity of larger windows, the benefits of the DIA-IT method are still superior to the potential limitations of low-resolution analyzers to resolve complex spectra.These results, together with recent reports on the use of ion trap-based parallel reaction monitoring using narrow windows, 24 confirm that the observed benefits of the linear ion trap do not strictly depend on the isolation scheme used (Supplementary Figure 4C).
Finally, given the low cycle time exhibited by the evaluated DIA-IT methods, we assessed whether we could dedicate some additional time to further improve the specificity of the DIA-IT methods without compromising their cycle time.Indeed, the inherent low resolution of linear ion traps limits their specificity, which is one of their main disadvantages when compared to high-resolution mass analyzers.For this purpose,  we set a DIA-IT method with a large number of small isolation windows, i.e., 100 windows of 4 Da covering the range of 500− 900 m/z, to improve the method specificity, minimize fragment ion interferences, and facilitate peptide identification.
We used this method to analyze the former serial dilutions of HeLa protein digest (1, 10, 100, and 1000 ng) and compared it to the previously evaluated DIA-IT and DIA-OT methods bearing an isolation scheme of 40 windows of 10 Da each.Our results showed a consistent increase in the number of identified peptides in all DIA-IT methods when using a large number of small isolation windows, independently of the scanning mode (normal, rapid, turbo) and the protein amount being analyzed (Figure 1G, Supplementary Figure 4D).Moreover, the higher specificity of DIA-IT 100X4 methods enable them to maintain a similar coefficient of variation in triplicate analyses than DIA-IT 40X10 methods, despite the increase in cycle time (Supplementary Figure 4E).Overall our results demonstrate the benefits of using linear ion trap-based DIA methodsespecially their high-specificity variant (100 windows of 4 Da each)to improve the number of identified peptides in low-input proteomes.−27 In conclusion, the use of linear ion trap-based DIA methods benefits peptide identifications in low-input proteomics applications, and together with recent analytical advances, they might further contribute to the feasibility of single cell proteomics.
The mass spectrometry proteomics data obtained from DIA-IT and all its companion methods have been deposited at the ProteomeXChange Consortium via the PRIDE repository with identifier PXD025766. 28

Figure 1 .
Figure 1.(A) Comparison of number of peptide ions identified among the reference DIA-OT method (DIA-IT 40X10 30K) and three DIA-IT 40X10 methods at different scanning speeds (normal, rapid, turbo) when analyzing 1 ng, 10 ng, 100 ng, and 1 μg of HeLa protein digest.(B) Comparison of the number of peptide ions identified when performing triplicate analyses of 1 ng of HeLa protein digest with the reference DIA-OT method (DIA-OT 40X10 30K) and three DIA-IT 40X10 methods at different scanning speeds (normal, rapid, turbo).(C) Overlap of the set of peptide sequences identified in triplicate analyses between the reference DIA-OT method (DIA-IT 40X10 30K) and DIA-IT 40X10 normal scan rate when analyzing 10 ng and 1 μg of HeLa protein digest.(D) Distribution of coefficient of variation of the DIA-OT 40X10 30K and the DIA-IT 40X10 normal methods when analyzing 10 ng and 1 μg of HeLa protein digest in triplicate.(E) Correlation of peptide abundances obtained from

Table 1 .
Main Parameters for Different DIA Methods Evaluated