Distinguishing and Quantifying Peptides and Proteins Containing<scp>d</scp>-Amino Acids by Tandem Mass Spectrometry

Adams, Christopher M.; Zubarev, Roman A.

doi:10.1021/ac0503963

Cited by 68 publications

(72 citation statements)

References 45 publications

(100 reference statements)

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“…one of the two methionines present). Potentially a structural modification like racemization of amino acids could cause a significant change in the elution time in nano-LC (40). If one takes into account the multiplicity of different isoforms of the dependent peptides, the N ab value increases to 7.8.…”

Section: Resultsmentioning

confidence: 99%

Extent of Modifications in Human Proteome Samples and Their Effect on Dynamic Range of Analysis in Shotgun Proteomics

Nielsen

Savitski

Zubarev

2006

Molecular & Cellular Proteomics

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The complexity of the human proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8 -12 modified peptides per each unmodified tryptic peptide present at >1% level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach. Molecular & Cellular Proteomics 5:2384 -2391, 2006.The human proteome is the most complex system in molecular biology. Today's consensus puts the number of human genes in the range from 20,000 to 25,000 (1). Further complexity is added at several levels mainly in the form of alternative splicing and post-translational modifications (PTMs).1 It is believed that at least 40 -60% of all human genes give alternative splicing isoforms (2, 3). Large scale studies on chromosomes 21 and 22 indicate that over 80% of the genes could undergo alternative splicing (4). Once synthesized on the ribosomes, most proteins undergo a multitude of PTMs, the exact type and position of which often cannot be predicted based on genetic information alone. These PTMs can consist of protein chains being cleaved, many different chemical groups can be attached to them (e.g. acetyl, methyl, phosphoryl, sugars, and lipids), and finally proteins can be internally or externally cross-linked by e.g. disulfide bonds. More than 200 different types of PTMs are currently known, and many more are yet to be discovered (5). With 20 common amino acids, this figure corresponds to more than 10 different types of PTM per amino acid. When combining the complexity generated by alternative splicing with that produced by PTMs, the current estimate of the number of different protein molecules expressed in a given individual organism is close to a million, which is roughly 50 forms per gene (6). Because an average human protein consists of 500 amino acids, the overall modification rate is approximately one modification per 10 amino acid residues. With all likel...

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Section: Resultsmentioning

confidence: 99%

Extent of Modifications in Human Proteome Samples and Their Effect on Dynamic Range of Analysis in Shotgun Proteomics

Nielsen

Savitski

Zubarev

2006

Molecular & Cellular Proteomics

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“…ECD holds much promise as a supplementary dissociation technique to CID for unambiguous protein identification, de novo sequencing (43) and detailed protein characterization (44). The method has been shown to distinguish leucine and isoleucine residues (45) and even differentiate between peptides containing a D-or L-amino acid within its sequence (46). ECD had also been applied to intact protein analysis (47) delivering a good level of fragmentation.…”

Section: Effect Of Magnetic Field On Resolution (High Field Magnets)-mentioning

confidence: 99%

Fourier Transform Mass Spectrometry

Ščigelová

Hornshaw

Giannakopulos

et al. 2011

Molecular & Cellular Proteomics

182

137

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Mass spectrometers have been used for a long time in a variety of biological applications. Recent years, however, have witnessed a significant increase in the employment of mass spectrometers such as of time-of-flight, Fourier transform ion cyclotron resonance (FTICR) 1 , and Orbitrap, which provide accurate mass of analytes over wide mass range. The FTICR and Orbitrap analyzers outperform any other commonly used mass spectrometer with respect to the maximum mass resolution and accuracy routinely achievable even for small numbers of ions. Both these mass spectrometers share certain features, such as an image current detection system and the application of Fourier transform mathematical operations for generating mass spectra from time domain transients produced by the image current. Consequently, they are often referred to as Fourier transform-based mass spectrometers (FTMS).The combination of FTMS with ion preselection and fragmentation devices and their coupling to reversed-phase liquid chromatography (LC) represents a ubiquitous approach to both small molecule and proteomic analyses. Multidimensional LC separations have an important role to play in proteomics applications for reducing sample complexity, and complement well the high dynamic range of detection in an acquisition offered by FTMS instruments.The current article does not intend to be an exhaustive review paper on FTMS techniques; it is intended as teaching material for scientists without a physics background to assist them with understanding the mass spectrometry tools they are called to use in their everyday laboratory work. Thus, only the fundamental aspects of FTMS which are useful to a nonphysicist are introduced. The discussion then focuses on examples of where this technology can be applied and what are the benefits as well as limitations of this technology for practical proteomic applications.Need for High Resolution and Mass Accuracy-The mass accuracy is the ratio of the m/z measurement error to the true m/z, usually quoted in parts per million (ppm). The mass resolving power (resolution) is the measure of the ability to distinguish two peaks of slightly different m/z, herein understood as full width at half maximum (FWHM). Linearity of detection and very high fidelity in the determination of frequency are inherent to FTMS and allow very high resolving power, mass accuracy, and dynamic range to be achieved. But why would one need high mass accuracy and high resolution?The benefit of measuring a compound's mass with adequately high accuracy can directly determine its elemental composition. Accurate mass thus acts as a powerful "filter" useful for confirming the identity of a compound or even identification of an unknown. This is illustrated in Fig. 1 showing the fragmentation (tandem mass spectrometry (MS/MS)) spectrum of flavonoid quercetin (m/z 303). Mass deviation of less than 3 ppm for any detected fragment together with the richness of the fragmentation spectra itself enable confirming the elemental composition of the starting compound a...

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“…Both electron capture dissociation (ECD) and CID have been used in the study of fragmentation of peptides containing damino acid substitutions [14,15]. According to these studies, it is the doubly charged protonated parent ions that exhibit the highest stereo chemical sensitivity [14].…”

Section: Discussionmentioning

confidence: 99%

“…According to these studies, it is the doubly charged protonated parent ions that exhibit the highest stereo chemical sensitivity [14]. The greatest impact of chiral sensitivity will then be expected for ions of low abundance, because these can be caused to enhance or to disappear from the product ion spectrum.…”

Section: Discussionmentioning

confidence: 99%

Use of synthetic analogues in confirmation of structure of the peptide antibiotics Maltacines

Hagelin¹,

Indrevoll

Høeg-Jensen

2007

International Journal of Mass Spectrometry

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Distinguishing and Quantifying Peptides and Proteins Containingd-Amino Acids by Tandem Mass Spectrometry

Cited by 68 publications

References 45 publications

Extent of Modifications in Human Proteome Samples and Their Effect on Dynamic Range of Analysis in Shotgun Proteomics

Extent of Modifications in Human Proteome Samples and Their Effect on Dynamic Range of Analysis in Shotgun Proteomics

Fourier Transform Mass Spectrometry

Use of synthetic analogues in confirmation of structure of the peptide antibiotics Maltacines

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