Tandem Mass Spectrometry

Tandem mass spectrometry, commonly known as tandem MS/MS is a mass spec- trometry comprised of two stages of the mass spectrometer. There are two mass analyzers in tandem MS/MS. The first mass analyzer is responsible for isolating ions of specific m/z values representing a particular peptide coming from the ion source. Tandem MS/MS breaks down precursor ions into product ions also known as frag- ment ions. The product ions are the ions that show the chemical composition of the precursor ions. Those ions successfully isolated by the first mass analyzer are acceler- ated into a collision cell chamber holding inert gas. This is where the fragmentation of the ions take place. The process is termed collision induced dissociation (CID) or collision activated dissociation (CAD). The second mass analyzer measures the m/z values of the fragmented ions to obatin the sequence of the peptide.

BioTechniques 40:790-798 (June 2006) doi 10.2144/000112201 Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecu- lar function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and bio- molecular structures.

Chapter 4 Glycan Structure Identification using Database Search and De Novo Sequencing Currently, two commonly used computational approaches for glycan structure identification with tandem mass spectrometry are database search and de novo sequencing. To some extent, de novo sequencing method is more di ffi cult than database search method, since it does not rely on any glycan database. Glycan identification by de novo sequencing can only rely on the information obtained from tandem mass spectra, without the assistance of any structural knowledge of glycans. Generally, database search method has the ability to obtain more re- liable results than de novo sequencing method. However, a distinctive advantage of glycan de novo sequencing is that it can identify some new glycan structures which are not included in the glycan database. With the development of mass spectrometry technology, more quality mass spectra will be produced, which can be used for glycan de novo sequencing to get more accurate results. Both of these two approaches have advantages and disadvantages, thus a bet- ter way is to combine them together for interpretation of glycan structures from tandem mass spectra.

T HE California legislature authorized and funded a pilot tandem mass spectrometry (MS/MS) screening program, which was conducted from January 7, 2002, through June 13, 2003. During that period, 755 698 infants were born, 353 894 newborns were screened, and 53 cases of disorders detectable with MS/MS were identified in the screened population (including 2 cases that were missed initially with MS/MS testing but would have been identified with our revised cutoff values). On the basis of this experience, the overall incidence in California is ⬃ 1 case per 6700 newborns screened. We did not include phenylketonuria (PKU) because we were evaluating the marginal benefits of MS/MS screening, compared with the incremental cost of adding the new technology to the existing screening program, which already screens for PKU. Using this incidence figure, we project that statewide screening of 540 000 annual births would, on average, identify 83 cases with disorders de- tectable through MS/MS screening.

Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose- β1,3- glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error. Electron transfer dissociation (ETD) preserves O-linked glycans on peptide fragments, but only a subset of tryptic peptides with low m/z can be reliably sequenced from ETD spectra compared to CID. Accordingly, studies to date that have used MS to identify O-fucosylated TSRs have required manual interpretation of CID mass spectra even when ETD was also employed. In order to facilitate high-throughput, automatic identification of O-fucosylated peptides from CID spectra, we re-engineered the MS/MS sequence database search engine Comet and the MS data analysis suite Trans-Proteomic Pipeline to enable automated sequencing of peptides exhibiting the neutral losses characteristic of labile O-linked glycans. We used our approach to re-analyze published proteomics data from Plasmodium parasites and identified multiple glycoforms of TSR-containing proteins.

29 Liebl B, Nennstiel-Ratzel U, von Kries R, Fingerhut R, Olgemoller B, Zapf A et al. Very high compliance in an expanded MS-MS-based newborn screening program despite written parental consent. Prev Med 2002;34:127-31. 30 Chace DH, Sherwin JE, Hillman SL, Lorey F, Cunningham GC. Use of phenylalanine-to- tyrosine ratio determined by tandem mass spectrometry to improve newborn screening for phenylketonuria of early discharge specimens collected in the first 24 hours. Clin Chem 1998;44:2405-9.

1 CHAPTER 1: LITERATURE REVIEW 1 Overview of Protein Identification Methods Many years ago, the Edman degradation was used to identify protein sequences. 1 This process involves the reaction of N terminus amino acid of a peptide with a chemical reagent that cleaves the amino acid from the peptide. The resultant compound is then used to identify the amino acids present in that peptide. This process involves many chemical reactions that can take several days and the sequenced peptide cannot be longer than 50-60 residues in length. In the early 90s, the identification of proteins and peptide was revolutionized by the use of mass spectrometry (MS). 2 The identification of proteins and peptides using MS is much more sensitive and faster because this technique uses the mass of amino acids residues and can fragment peptides in seconds rather than in days. Proteins are often digested with trypsin enzyme and the resulting peptides are further subject to MS analysis. The amino acids sequence can be determined with tandem mass spectrometry (MS/MS) which uses Collision Induced Dissociation (CID) to further fragment peptides. 3

Conclusions A rapid and simple FIA-MS/MS method for accurate quantitation of creatinine in human urine was developed. Isotope dilution tandem mass spectrometry with quali- fier ion monitoring ensured unbiased quantitation and qualification of creatinine in every single sample. Preci- sion and accuracy were in accordance with widely accept- ed bioanalytical standard recommendations and mostly superior to previously published protocols. Comparison with results measured by the colorimetric Jaffé assay rou- tinely applied in clinical chemistry showed high agree- ment. Being characterized by a wide linear calibration range and excellent accuracy, the assay is applicable for the full range of creatinine concentrations observed in human urine. The method offers the advantages of low required sample volume as well as straightforward and fast sample preparation. Feasible throughput of more than 1,400 injections per day at low costs per sample makes it an attractive choice for creatinine determina- tions in large-scale epidemiological or clinical studies.

2 School of Medicine, University of Maryland Baltimore, Baltimore, MD, USA Abstract Introduction—Endodontic infections are very prevalent and have a polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and the host defenses. Proteomic analysis of endodontic infections can provide global insights into the invasion, pathogenicity mechanisms, and multifactorial interactions existing between root canal bacteria and the host in the initiation and progression of apical periodontitis. The purpose of this study was to apply proteomic techniques such as liquid chromatography–tandem mass spectrometry (LC–MS/

Sample solutions were infused directly into an electrospray ionisation (ESI) source at 5 μL min 1 , from which positive ions were chosen for analysis. A heated capillary was set to 350°C and a sheath gas (nitrogen) optimised to a flow rate that delivered a stable spray. The spray voltage was 4 kV and the tube lens voltage was optimised to give the most intense precursor ion signal. For tandem mass spectrometry the isolation window was set 4 u. CID was performed in the ion trap region of this hybrid instrument using helium as the collision gas with 20–30 eV normalised collision energy, with the product ions measured in the FTICR instrument. For EID, the indirectly heated dispenser cathode inside the FTICR cell generated electrons at 26.5 eV for 70 ms. All data were recorded and processed using Xcalibur software (version 2.0;

Chapter 8 Conclusions and Future Directions 8.1 Summary The work described in the previous chapters involved the development of methods aimed at improving the information content from tandem mass spectrometry experiments. The use of IAM on a Q-FTICR-MS instrument was shown to increase the number of parent ions that could be studied using MS/MS over a given amount of time. The operation of ECD on a Q-FTICR-MS and a ECD LIT /TOF was compared. Results demonstrated that the information provided by ECD on both instruments is very similar, suggesting that the benefits of ECD can be realized on non-FTICR mass spectrometers. A new method, referred to as ECD+CID, was developed to improve the quality of the data acquired from the ECD LIT instrument. The first examples of EDD and AI-ECD performed in a LIT were also characterized using the ECD LIT . All of these topics have shown promise for improving MS/MS analyses. The results of these studies are summarized here and potential areas for continued research are discussed.

All mass spectrometric measurements were performed on a hybrid linear ion trap–Fourier transform-ion cyclotron mass spectrometer equipped with a 7.0 T superconducting magnet (LTQFT from ThermoFinnigan, Bremen, Germany). Sample solutions were infused directly into an electrospray ionisation (ESI) source at 5 μL min 1 , from which positive ions were chosen for analysis. A heated capillary was set to 350°C and a sheath gas (nitrogen) optimised to a flow rate that delivered a stable spray. The spray voltage was 4 kV and the tube lens voltage was optimised to give the most intense precursor ion signal. For tandem mass spectrometry the isolation window was set 4 u. CID was performed in the ion trap region of this hybrid instrument using helium as the collision gas with 20–30 eV normalised collision energy, with the product ions measured in the FTICR instrument. For EID, the indirectly heated dispenser cathode inside the FTICR cell generated electrons at 26.5 eV for 70 ms. All data were recorded and processed using Xcalibur software (version 2.0; ThermoFinnigan, San Jose, CA, USA). Data were internally calibrated from the precursor ion and the common product ion [H 4 PO 4 ] + .

Phosphorylated ions have predictable vibrational tandem mass spectrometric behaviour, with loss of phosphate groups predominantly observed owing to the labile nature of the bond. [30–34] Cyclic adenosine monophosphate (cAMP) analogues have been studied by CID, with limited product ions formed. The majority of product ions related to cleavage between the ribose-phosphate and aminopurine sub- structures. [30] The limited nature of the product ions formed from CID for this type of ion has led to studies aimed at gaining increased information from tandem mass spectrometry, including the use of electron-based techniques. ECD of adenosine triphosphate (ATP) and analogous ions was shown to yield complementary product ions to CID for doubly charged precursor ions. Unique to ECD, ribose cross- ring cleavage was observed as well as several hydrated product ions formed by cross-ring cleavages at other groups. As CID also yields unique product ions, Liu et al. concluded that a combination of ECD and CID can be implemented to gain maximum product ion information for acidic metabolites. [35] ECD also results in high sequence coverage for phosphopeptides containing up to four phosphorylation sites. [31] The effect of increasing the number phosphate groups has on sequence coverage by ECD was that for [M + 2H] 2+ , increased levels of phosphorylation results in lower sequence coverage of the peptides. This is thought to be caused by the phosphate groups forming salt bridges with amino acid side

limiting result of increasing the time required for a single chromatographic separation Mass spectrometry is generally thought to be much less time consuming than HPLC when only a single dimension of mass analysis is required. However, mass spectrometry will also suffer from the deleterious effects of increased analysis time when tandem mass spectrometry (MS/MS) or multiple stages of mass spectrometry (MSn) are implemented. MS/MS is the essential step in sequencing peptides for bottom-up proteomics. The process of MS/MS requires isolation of a parent prior to its activation. Isolation of the parent ion is necessary for the genealogy of the parent ion to be preserved. Without knowledge of a product ion’s parent ion, interpretation of a MS/MS spectrum is a laborious and near impossible task.

Peter Mollee 1* , Samuel Boros 2 , Dorothy Loo 3 , Jayde E. Ruelcke 3 , Vanessa A. Lakis 3 , Kim‑Anh Lê Cao 3 , Patricia Renaut 2 and Michelle M. Hill 3* Abstract Background: Correct identification of the amyloidosis‑causing protein is crucial for clinical management. Recently the Mayo Clinic reported laser‑capture microdissection (LCM) with liquid chromatography‑coupled tandem mass spectrometry (MS/MS) as a new diagnostic tool for amyloid diagnosis. Here, we report an independent implemen‑

Nawed IK Deshmukh 1* , James Barker 1 , Andrea Petroczi 2 and Declan P Naughton 2 Abstract Background: Owing to frequent administration of a wide range of pharmaceutical products, various environmental waters have been found to be contaminated with pharmacologically active substances. For example, stanozolol, a synthetic anabolic steroid, is frequently misused for performance enhancement as well as for illegal growth promoting purposes in veterinary practice. Previously we reported stanozolol in hair samples collected from subjects living in Budapest. For this reason we initiated this study to explore possible environmental sources of steroid contamination. The aim of this study was to develop a method to monitor stanozolol in aqueous matrices using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Keywords Insulin, Tandem Mass Spectrometry, Electrospray, de novo Sequencing 1. Introduction Diabetes is currently an incurable and potentially life-threatening disorder that occurs in patients whose pancreas β cells cannot secrete a sufficient amount of insulin into the blood, resulting in elevated blood glucose levels. In severe cases, the gland could lose its function entirely. High blood sugar levels cause severe impairments in all the body’s organs, particularly the kidneys, the nervous sys-

ABSTRACT A simple and sensitive method for the determination of pyrethroid pesticide residues namely transfluthrin, allethrin, bifenthrin, lambda cyhalothrin, permethrin, cyfluthrin, cypermethrin, ethofenprox, fenvalerate, tauflivalinate & deltamethrin in rice grains by gas chromatography –tandem mass spectrometry (GC-MS/MS) was developed. The pyrethroid residues were extracted from homogenized rice samples initially with water followed by acetonitrile and cleaned up by using dispersive solid phase extraction which is based on QuEChERS (quick, easy, cheap, rugged and safe) method, finally the pyrethroid pesticide residues were separated by DB-5MS gas chromatography capillary column and quantified through tandem mass spectrometry (GC-MS/MS) with electron ionization source in Multiple reaction monitor mode (MRM) for quantification and confirmation. Recoveries were checked at two fortification levels 0.01 and 0.05mg/kg (n=6). The results showed that the mean recoveries for the fortified samples were ranged between 87-117% and %RSD in the range between 5.9 -19.8 for all compounds. Thus the developed analytical method was successfully used to analyze pyrethroid pesticide residues in routine rice samples .

Hybrid MS/MS instruments use various combina- tions of these analyzers to obtain desirable performance characteristics for the phenomena being investigated. Typically, resolving power and measurement accuracy in each stage of the mass spectral analysis are of primary importance. However, the kinetic energy used for CID can also be a critical parameter, depending on the information being sought. Additionally, the speed of analysis (duty cycle) can also be an important param- eter. The development of many early hybrid instru- ments was motivated by the desire for fast MS/MS spectral acquisition times required by the time scale of a chromatographic separation. The early hybrid instru- ments (1980s) all used beam type analyzers (beam-beam instruments) because these were the standards of the day, with trapping instruments just being introduced as commercially viable mass analyzers. Two of these beam-beam type hybrid instruments evolved into com- mercial instruments, sector/Q and Q/TOF, with the latter one still being a popular instrument today. Sub- sequently (1990s), beam-trap type instruments were explored, followed by trap-beam instruments. A hand- ful of beam-trap instruments (sector/QIT) have been commercialized. The trap-beam instrument closely re- lated to the Q/TOF, the QIT/TOF, has become commer- cially available. Most recently (2000s), trap-trap type hy- brid instruments have been commercially developed. Not surprisingly, a general trend in the evolution of hybrid instruments is that their development is driven by ad-

Surface induced dissociation (SID) spectra of peptides were obtained by means of a four sector mass spectrometer with a modified collision cell in the third field free regi[r]