Improved Precursor Characterization for Data-Dependent Mass Spectrometry.

Modern ion trap mass spectrometers are capable of collecting up to 60 tandem MS (MS/MS) scans per second, in theory providing acquisition speeds that can sample every eluting peptide precursor presented to the MS system. In practice, however, the precursor sampling capacity enabled by these ultrafast acquisition rates is often underutilized due to a host of reasons (e.g., long injection times and wide analyzer mass ranges). One often overlooked reason for this underutilization is that the instrument exhausts all the peptide features it identifies as suitable for MS/MS fragmentation. Highly abundant features can prevent annotation of lower abundance precursor ions that occupy similar mass-to-charge (m/z) space, which ultimately inhibits the acquisition of an MS/MS event. Here, we present an advanced peak determination (APD) algorithm that uses an iterative approach to annotate densely populated m/z regions to increase the number of peptides sampled during data-dependent LC-MS/MS analyses. The APD algorithm enables nearly full utilization of the sampling capacity of a quadrupole-Orbitrap-linear ion trap MS system, which yields up to a 40% increase in unique peptide identifications from whole cell HeLa lysates (approximately 53 000 in a 90 min LC-MS/MS analysis). The APD algorithm maintains improved peptide and protein identifications across several modes of proteomic data acquisition, including varying gradient lengths, different degrees of prefractionation, peptides derived from multiple proteases, and phosphoproteomic analyses. Additionally, the use of APD increases the number of peptides characterized per protein, providing improved protein quantification. In all, the APD algorithm increases the number of detectable peptide features, which maximizes utilization of the high MS/MS capacities and significantly improves sampling depth and identifications in proteomic experiments.

Multiplexed MS/MS for improved data-independent acquisition.

In mass spectrometry-based proteomics, data-independent acquisition (DIA) strategies can acquire a single data set useful for both identification and quantification of detectable peptides in a complex mixture. However, DIA data are noisy owing to a typical five- to tenfold reduction in precursor selectivity compared to data obtained with data-dependent acquisition or selected reaction monitoring. We demonstrate a multiplexing strategy, MSX, for DIA analysis that increases precursor selectivity fivefold.

The Q Exactive HF, a Benchtop mass spectrometer with a pre-filter, high-performance quadrupole and an ultra-high-field Orbitrap analyzer.

The quadrupole Orbitrap mass spectrometer (Q Exactive) made a powerful proteomics instrument available in a benchtop format. It significantly boosted the number of proteins analyzable per hour and has now evolved into a proteomics analysis workhorse for many laboratories. Here we describe the Q Exactive Plus and Q Exactive HF mass spectrometers, which feature several innovations in comparison to the original Q Exactive instrument. A low-resolution pre-filter has been implemented within the injection flatapole, preventing unwanted ions from entering deep into the system, and thereby increasing its robustness. A new segmented quadrupole, with higher fidelity of isolation efficiency over a wide range of isolation windows, provides an almost 2-fold improvement of transmission at narrow isolation widths. Additionally, the Q Exactive HF has a compact Orbitrap analyzer, leading to higher field strength and almost doubling the resolution at the same transient times. With its very fast isolation and fragmentation capabilities, the instrument achieves overall cycle times of 1 s for a top 15 to 20 higher energy collisional dissociation method. We demonstrate the identification of 5000 proteins in standard 90-min gradients of tryptic digests of mammalian cell lysate, an increase of over 40% for detected peptides and over 20% for detected proteins. Additionally, we tested the instrument on peptide phosphorylation enriched samples, for which an improvement of up to 60% class I sites was observed.

Myocardial blood flow in patients with transposition of the great arteries ­ risk factor for dysfunction of the morphologic systemic right ventricle late after atrial repair.

BACKGROUND: Dysfunction of the morphologic systemic right ventricle (RV) is a sequela in long-term survivors with transposition of the great arteries (TGA) after atrial switch operation (AtSO). Impairment of myocardial blood flow (MBF) and coronary flow reserve (CFR) are hypothesized as predisposing factors. METHODS AND RESULTS: The study group comprised 20 patients after AtSO (22.7 ± 5.03 years) and 15 individuals with congenitally corrected transposition (ccTGA) (30.6 ± 19.4 years). MBF was quantified by positron emission tomography; controls for coronary flow were 11 healthy volunteers (26.2 ± 5.1 years). Exercise capacity, ventricular mass, function and end-diastolic volume assessed by coronary magnetic resonance (CMR), hemodynamic parameters assessed by cardiac catheterization and echocardiography, and B-type natriuretic peptide levels correlated with MBF. At rest, MBF did not differ between patients and healthy volunteers (MBFrestml·100 g(-1)·min(-1); ccTGA: 75 ± 14 vs. AtSO: 73 ± 16 vs. controls: 77 ± 15; NS). After vasodilatation, MBF increased significantly, but was significantly lower in ccTGA and AtSO groups compared with controls (MBFstressml·100 g(-1)·min(-1); ccTGA: 198 ± 38 vs. AtSO: 167 ± 46 vs. controls 310 ± 74; P<0.001). In ccTGA, CFR correlated significantly with clinical, CMR, echocardiographic and hemodynamic parameters, but for AtSO patients no significant correlation could be calculated. CONCLUSIONS: In patients with ccTGA, maximal coronary blood flow is attenuated and significantly correlated with ventricular function, whereas dysfunction of the morphologic systemic RV after AtSO is a multifactorial problem.

Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.

Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate the faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange data set PXD001305). We find the ultra-high-field Orbitrap mass analyzer to be capable of attaining a sequencing speed above 20 Hz, and it routinely exceeds 10 peptide spectrum matches per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from 1 µg of HeLa digest using a 1 h gradient, which is an approximately 30% improvement compared to that with previous instrumentation. In addition, we show that very deep proteome coverage can be achieved in less than 24 h of analysis time by offline high-pH reversed-phase peptide fractionation, from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multiday, multienzyme efforts. Finally, the acquisition methods are evaluated for single-shot phosphoproteomics, where we identify 7600 unique HeLa phosphopeptides in one gradient hour and find the quality of fragmentation spectra to be more important than quantity for accurate site assignment.

Ventricular sympathetic innervation in patients with transposition of the great arteries after arterial switch operation and Rastelli procedure: impact of arterial dissection and coronary reimplantation.

BACKGROUND: Coronary flow reserve (CFR) is reduced in patients with transposition of the great arteries (TGA) after the arterial switch operation (ASO). Dissection of the great arteries and coronary reimplantation may result in sympathetic denervation, with a negative effect on myocardial perfusion. METHODS AND RESULTS: 18 patients with TGA participated in the study; 9 had ASO (20.8±5.8 years). Controls were 9 patients after Rastelli procedure (22.1±6.8 years). Sympathetic innervation was measured by positron emission tomography using(11)C epinephrine (EPI). Left ventricular EPI-retention ranged from 6.1% to 15.9%/min. Patients undergoing more than 1 operation had significantly reduced EPI-retention (P<0.001). EPI-retention and time interval after surgery correlated significantly (r=0.81, P<0.001) and was higher in patients undergoing surgery at an earlier age (P<0.001). No significant difference could be found between patients after ASO or Rastelli repair. Aortic cross-clamp time inversely correlated with EPI-retention (r=-0.72; P<0.001). CONCLUSIONS: The ASO procedure had a negative effect on sympathetic innervation of the myocardium, but because of reinnervation myocardial perfusion is not essentially altered by this mechanism. Heart surgery and prolonged aortic cross-clamp time have a negative effect on the norepinephrine content of cardiac sympathetic nerve terminals. Parameters such as ventricular performance and cardiopulmonary exercise capacity were unaffected by the degree of EPI-retention.

Managing the right ventricular outflow tract for pulmonary regurgitation after tetralogy of Fallot repair.

The long-term outcome of patients with tetralogy of Fallot (TOF) with reconstruction of the right ventricular (RV) outflow tract is often complicated by the sequelae of severe pulmonary regurgitation. Progressive enlargement of the right ventricle, biventricular dysfunction and arrhythmia are apparent in more than 50% of the patients in the fourth decade of life. Pathophysiologic implications, clinical assessment and diagnostic modalities are discussed, whereas CMR imaging seems to be the procedure of choice. Therapeutical options for rereconstruction of the RV outflow tract are mentioned, surgical and interventional procedures are explained in detail. The optimal timing of reoperation for significant pulmonary regurgitation after TOF repair is still a matter of controversy given the limited runtime of the lately implanted prostheses and the risk of further reoperation. Early surgery is recommended in these patients before symptoms develop, or RV function has declined. Today we believe that waiting for the patient to become symptomatic is too late. All in all, pulmonary valve replacement is at least indicated in patients developing symptoms due to severe pulmonary regurgitation, particularly if associated with substantial or progressive RV dilatation, tricuspid regurgitation and/or supraventricular or ventricular arrhythmias.