A Causal Model of Ion Interference Enables Assessment and Correction of Ratio Compression in Multiplex Proteomics.

Multiplex proteomics using isobaric labeling tags has emerged as a powerful tool for the simultaneous relative quantification of peptides and proteins across multiple experimental conditions. However, the quantitative accuracy of the approach is largely compromised by ion interference, a phenomenon that causes fold changes to appear compressed. The degree of compression is generally unknown, and the contributing factors are poorly understood. In this study, we thoroughly characterized ion interference at the MS2 level using a defined two-proteome experimental system with known ground-truth. We discovered remarkably poor agreement between the apparent precursor purity in the isolation window and the actual level of observed reporter ion interference in MS2 scans-a discrepancy that we found resolved by considering cofragmentation of peptide ions hidden within the spectral "noise" of the MS1 isolation window. To address this issue, we developed a regression modeling strategy to accurately predict reporter ion interference in any dataset. Finally, we demonstrate the utility of our procedure for improved fold change estimation and unbiased PTM site-to-protein normalization. All computational tools and code required to apply this method to any MS2 TMT dataset are documented and freely available.

TMT29 plex: Higher order multiplexing with inherent interference correction.

Tandem mass tag (TMT) technology enables mass spectrometry-based multiplexed sample profiling of protein abundance. The degree of isobaric labeling with TMT has increased from 2 to 6, 11, 16 [1] and recently 18 [2] channels, enabling the development of higher throughput assays. In TMT experiments, measurement of reporter tag intensities is confounded by ratio compression. Although both pre- and post-acquisition methods have been developed to decrease interference, ratio compression remains a problem in complex samples. The work of Sun et al. presented a TMT29-plex workflow that combines the TMT11-plex and TMTpro18-plex labeling strategies and uses their inherent features to address the ratio compression problem, increasing the TMT throughput and quantitative accuracy for potential applications [3].

29-Plex tandem mass tag mass spectrometry enabling accurate quantification by interference correction.

Tandem mass tag (TMT) mass spectrometry is a mainstream isobaric chemical labeling strategy for profiling proteomes. Here we present a 29-plex TMT method to combine the 11-plex and 18-plex labeling strategies. The 29-plex method was examined with a pooled sample composed of 1×, 3×, and 10× Escherichia coli peptides with 100× human background peptides, which generated two E. coli datasets (TMT11 and TMT18), displaying the distorted ratios of 1.0:1.7:4.2 and 1.0:1.8:4.9, respectively. This ratio compression from the expected 1:3:10 ratios was caused by co-isolated TMT-labeled ions (i.e., noise). Interestingly, the mixture of two TMT sets produced MS/MS spectra with unique features for the noise detection: (i) in TMT11-labeled spectra, TMT18-specific reporter ions (e.g., 135N) were shown as the noise; (ii) in TMT18-labeled spectra, the TMT11/TMT18-shared reporter ions (e.g., 131C) typically exhibited higher intensities than TMT18-specific reporter ions, due to contaminated TMT11-labeled ions in these shared channels. We further estimated the noise levels contributed by both TMT11- and TMT18-labeled peptides, and corrected reporter ion intensities in every spectrum. Finally, the anticipated 1:3:10 ratios were largely restored. This strategy was also validated using another 29-plex sample with 1:5 ratios. Thus the 29-plex method expands the TMT throughput and enhances the quantitative accuracy.

Evaluation of a Dual Isolation Width Acquisition Method for Isobaric Labeling Ratio Decompression.

Isobaric labeling is a highly precise approach for protein quantification. However, due to the isolation interference problem, isobaric tagging suffers from ratio underestimation at the MS2 level. The use of narrow isolation widths is a rational approach to alleviate the interference problem; however, this approach compromises proteome coverage. We reasoned that although a very narrow isolation window will result in loss of peptide fragment ions, the reporter ion signals will be retained for a significant portion of the spectra. On the basis of this assumption, we have designed a dual isolation width acquisition (DIWA) method, in which each precursor is first fragmented with HCD using a standard isolation width for peptide identification and preliminary quantification, followed by a second MS2 HCD scan using a much narrower isolation width for the acquisition of quantitative spectra with reduced interference. We leverage the quantification obtained by the "narrow" scans to build linear regression models and apply these to decompress the fold-changes measured at the "standard" scans. We evaluate the DIWA approach using a nested two species/gene knockout TMT-6plex experimental design and discuss the perspectives of this approach.

Evaluation of Advanced Precursor Determination for Tandem Mass Tag (TMT)-Based Quantitative Proteomics across Instrument Platforms.

Tandem mass tag (TMT)-based quantitation is a strong modality for quantitative proteomics, as samples can be multiplexed, creating large-scale data sets with high precision and minimal missing values. However, coisolation/cofragmentation of near isobaric, coeluting precursor peptide analytes has been well-documented to show ratio compression, compromising the accuracy of peptide/protein quantitation. Advanced peak determination (APD) is a new peak-picking algorithm that shows improved identification of peak detection in survey scans (MS1) to increase the number of precursors selected for unimolecular dissociation (MS2). To increase the number of these "features" selected for MS2 APD purposefully selects multiple peptide precursors of very similar m/ z that often derive from different proteins-a major source of ratio compression in TMT quantification. Here, we evaluate the effects of various data acquisition parameters combined with APD on ratio compression. We find that data acquisition with APD enabled results in more coisolated precursors, more mixed spectra, and in turn, fewer peptide spectral matches, especially at standard on-column loads. We conclude that APD should not be utilized for isobaric tagging, MS2-based experiments.

Compositional Proteomics: Effects of Spatial Constraints on Protein Quantification Utilizing Isobaric Tags.

Mass spectrometry (MS) has become an accessible tool for whole proteome quantitation with the ability to characterize protein expression across thousands of proteins within a single experiment. A subset of MS quantification methods (e.g., SILAC and label-free) monitor the relative intensity of intact peptides, where thousands of measurements can be made from a single mass spectrum. An alternative approach, isobaric labeling, enables precise quantification of multiple samples simultaneously through unique and sample specific mass reporter ions. Consequently, in a single scan, the quantitative signal comes from a limited number of spectral features (≤11). The signal observed for these features is constrained by automatic gain control, forcing codependence of concurrent signals. The study of constrained outcomes primarily belongs to the field of compositional data analysis. We show experimentally that isobaric tag proteomics data are inherently compositional and highlight the implications for data analysis and interpretation. We present a new statistical model and accompanying software that improves estimation accuracy and the ability to detect changes in protein abundance. Finally, we demonstrate a unique compositional effect on proteins with infinite changes. We conclude that many infinite changes will appear small and that the magnitude of these estimates is highly dependent on experimental design.

On the Statistical Significance of Compressed Ratios in Isobaric Labeling: A Cross-Platform Comparison.

Isobaric labeling is gaining popularity in proteomics due to its multiplexing capacity. However, copeptide fragmentation introduces a bias that undermines its accuracy. Several strategies have been shown to partially and, in some cases, completely solve this issue. However, it is still not clear how ratio compression affects the ability to identify a protein's change of abundance as statistically significant. Here, by using the "two proteomes" approach (E. coli lysates with fixed 2.5 ratios in the presence or absence of human lysates acting as the background interference) and manipulating isolation width values, we were able to model isobaric data with different levels of accuracy and precision in three types of mass spectrometers: LTQ Orbitrap Velos, Impact, and Q Exactive. We determined the influence of these variables on the statistical significance of the distorted ratios and compared them to the ratios measured without impurities. Our results confirm previous findings1-4 regarding the importance of optimizing acquisition parameters in each instrument in order to minimize interference without compromising precision and identification. We also show that, under these experimental conditions, the inclusion of a second replicate increases statistical sensitivity 2-3-fold and counterbalances to a large extent the issue of ratio compression.

Evaluation of Protein Identification and Quantification by the diaPASEF Method on timsTOF SCP.

Accurate and precise quantification is crucial in modern proteomics, particularly in the context of exploring low-amount samples. While the innovative 4D-data-independent acquisition (DIA) quantitative proteomics facilitated by timsTOF mass spectrometers gives enhanced sensitivity and selectivity for protein identification, the diaPASEF (parallel accumulation-serial fragmentation combined with data-independent acquisition) parameters have not been systematically optimized, and a comprehensive evaluation of the quantification is currently lacking. In this study, we conducted a thorough optimization of key parameters on a timsTOF SCP instrument, including sample loading amount (50 ng), ramp/accumulation time (140 ms), isolation window width (20 m/z), and gradient time (60 min). To further improve the identification of proteins in low-amount samples, we utilized different column settings and introduced 0.02% n-dodecyl-ß-d-maltoside (DDM) in the sample reconstitution solution, resulting in a remarkable 19-fold increase in protein identification at the single-cell-equivalent level. Moreover, a comprehensive comparison of protein quantification using a tandem mass tag reporter (TMT-reporter), complement TMT ions (TMTc), and diaPASEF revealed a strong correlation between these methods. Both diaPASEF and TMTc have effectively addressed the issue of ratio compression, highlighting the diaPASEF method's effectiveness in achieving accurate quantification data compared to TMT reporter quantification. Additionally, an in-depth analysis of in-group variation positioned diaPASEF between the TMT-reporter and TMTc methods. Therefore, diaPASEF quantification on the timsTOF SCP instrument emerges as a precise and accurate methodology for quantitative proteomics, especially for samples with small amounts.

Chest Compressions During Sustained Inflation During Cardiopulmonary Resuscitation in Newborn Infants Translating Evidence From Animal Studies to the Bedside.

Newborn infants receiving chest compressions in the delivery room have a high incidence of mortality (41%) and short-term neurological morbidity (e.g., 57% hypoxic-ischemic encephalopathy and seizures). Furthermore, infants who have no signs of life at 10 min despite chest compressions have 83% mortality, with 93% of survivors experiencing moderate-to-severe disability. The poor prognosis associated with receiving chest compressions in the delivery room raises questions as to whether improved cardiopulmonary resuscitation methods specifically tailored to the newborn could improve outcomes. Combining chest compressions during sustained inflation (CC+SI) has recently been shown to improve morbidity and mortality outcomes during cardiopulmonary resuscitation. Overall, CC+SI accomplishes the following: 1) significantly reduces time to return of spontaneous circulation, mortality, and epinephrine administration, and improves systemic and regional hemodynamic recovery; 2) significantly increases tidal volume and minute ventilation, and therefore alveolar oxygen delivery; 3) allows for passive ventilation during chest compression; and 4) does not increase lung or brain injury markers compared with the current standard of using 3:1 compression:ventilation ratio. A randomized trial comparing CC+SI versus a 3:1 compression:ventilation ratio during cardiopulmonary resuscitation in the delivery room is therefore warranted.