Electrospray nanospray

Hybrid linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, 7T (LTQ-FTICR,Thermo), or equivalent instrument, equipped with an electrospray/nanospray (ESI/ NSI) source and ECD accessory, operated under the control of Xcalibur software package (Thermo Fisher Scientific, San Jose, CA Instrument Configuration, Instrument Setup, Sequence Setup, and Qual Browser modules appropriately created and used) with Bioworks available to preprocess raw data files for database search. ECD may be replaced with ETD and the respective manufacturer s own version of the above software should be used, when alternative instrument is selected. 

Nanoelectrospray and Insights into Fundamentals of Electrospray—Nanospray References... 

Nanoelectrospray has proven to be of enormous importance to the analysis of biochemical and biopharmaceutical samples. However, it is also important to research on the fundamentals of electrospray—nanospray. Karas and co-workers have been major contributors to this research. The experimental finding that mass spectra of analytes such as proteins obtained with nanospray are much less affected by the presence of impurities in the solution such as sodium compared to electrospray spectra is an advantage of nanospray. The reasons for this were examined and the following reason was given. Gas-phase ions are produced from charged droplets only when the droplets are very small. This holds for both IBM and CRM. Therefore, if one starts with relatively small initial droplets, as is the case with nanospray, much less solvent evaporation will be required to reach the small size droplets required. Therefore, in the presence of impurities such as sodium salts the concentration increase of the salt will be much smaller with nanospray. 

The HPLC system comprised a 75 ftm x 15 cm PepMap column with a linear gradient of acetonitrile/0.1% aqueous formic acid (5 to 50% acetonitrile over 45 min) at a flow rate of 250 nlmin . Positive-ion electrospray ionization was employed using a nanospray interface. MS-MS specna were acquired over the range m/z 40 to 2000 at a rate of 1 s per scan. 

Wetterhall, M., Nilsson, S., Markides, K. E., and Bergquist, J. (2002). A conductive polymeric material used for nanospray needle and low-flow sheathless electrospray ionization applications. Anal. Chem. 74, 239-245. 

Nanospray is a miniaturized version of electrospray. In the original setup of Wilm and Mann (8) it is utilized as an off-line technique using disposable, finely drawn (1 -gm tip), metallized glass capillaries to infuse samples at 10-30 nL/min flow rates. This allows more than 50 min analysis time with just a 1-pT sample. Due to the formation of much smaller droplets and the more effective ionization, there is often no need for LC separation, since the separation is accomplished in m/z or by MS/MS. However, limited reproducibility with respect to quantification and a more complex sample preparation can be seen as drawbacks. An on-line version for hyphenation with capillary and nano-LC as well as CE (slightly modified) is now commercially available. 

Nanoelectrospray ionization (nanoESI), also known as nanospray, nanoflow electrospray, and micro-electrospray, is a low flow/high sensitivity approach to ESI. NanoESI15 is a slight variation on ESI such that the spray needle has been made very small and is positioned close to the entrance of the vacuum of the mass spectrometer and the mass analyzer (Figure 6). This greatly reduces required sample amounts allowing nanoliter flow rates and femto-mole sample consumption. The end result is increased efficiency since the flow rates for... 

Figure 14.5 Modified-ESI source for the direct infusion of undiluted ILs. A stainless steel wire is placed in the spray, leading to the optimal vaporization of the IL. Additionally, an orthogonal ESI source is used. Only a part of the IL ions is transferred into the MS, thus minimizing pollution of the source. (Modified from Dyson, R J. et al.. Direct probe electrospray (and nanospray) ionization mass spectrometry of neat ionic liquids. Chem. Commun., 2204, 2004. Reproduced by permission of the Royal Society of Chemistry.)...

Dyson, P. J. et al.. Direct probe electrospray (and nanospray) ionization mass spectrometry of neat ionic liquids. Chem. Commun., 2204, 2004. 

Miniaturization of HPLC-ICP-MS is an important issue in bioanalytical chemistry when small amounts of sample (e.g., single cells) need to be investigated.33 ICP-MS (with an octopole collision cell) in combination with nano-HPLC (75 pun column) was optimized for the detection of selenopeptides in a selenium-yeast protein digest after 100-fold preconcentration on a C18 capillary precolumn (300 (im column for salt removal and cleanup).34 Under identical separation and preconcentration conditions, electrospray MS/MS (using Nanospray qQqToF-MS - QSTAR from Applied... 

Today, the two most common LC/MS interfaces are atmospheric pressure ionization interfaces, electrospray (ESI) and ion spray (ISI). Electrospray (Fig. 15.8) and its subtype, nanospray, are recommended for use with proteins and highly polar or ionized compounds. They are very soft ionization, concentration-dependent techniques that result in very little fragmentation and often produce multiply charged molecular ions. 

Two factors are driving the market for precise, very-low-flow HPLC pumping systems extremely limited sample sizes in biotechnology and the electrospray and nanospray interfaces that are concentration and flow-rate dependent. It is very difficult to get precise flow and gradient formation from pumps that have a 5- to 10-/iL plunger displacement, even using 3200-step stepper motor drives. This has forced manufacturers to resurrect a very old concept from the earliest days of HPLC, the syringe pump. 

Nanoflow HPLC—HPLC system with accurately controlled reciprocating and syringe pumps designed to use capillary and small diameter, high-resolution columns as front ends for electrospray and nanospray mass spectrometer interfaces. 

Gatlin, C. L. Kleemann, G. R. Hays, L. G. Link, A. J. Yates, J. R. 1998. Protein identification at the low femtomole level from silver-stained gels using a fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal. Biochem., 263, 93-101. 

For all of the above good reasons, few publications have come out on combinatorial mixture analysis in this review period. Mixture analysis of peptides by MS and MS/MS has continued as an active field [2,59,64], Nanospray (very low flow electrospray) has become the method of choice for studies of mixtures of peptides by MS/MS [65,66], 2D NMR has been used to assure that unusual amino acids were correctly incorporated in a mixed synthesis [59]. 

Figure 31 Schematic diagram of the nanospray tip for the electrospray source. (Reprinted with permission from Ref 435a. 2003 American Chemical Society)...

Guaratini T, Gates PJ, Cardozo KH, Campos PM, Colepicolo P, Lopes NP. Letter Radical ion and protonated molecule formation with retinal in electrospray and nanospray. Eur J Mass Spectrom 2006 12 71-74. 

The nano-electrospray (nanoES) source is essentially a miniaturized version of the ES source. This technique allows very small amounts of sample to be ionized efficiently at nanoliters per minute flow rates and it involves loading sample volumes of 1-2 pi into a gold-coated capillary needle, which is introduced to the ion source. Alternatively for on-line nanoLC-MS experiments the end of the nanoLC column serves as the nanospray needle. The nanoES source disperses the liquid analyte entirely by electrostatic means [27] and does not require assistance such as solvent pumps or nebulizing gas. This improves sample desolvation and ionization and sample loading can be made to last 30 minutes or more. Also, the creation of nanodroplets means a high surface area to volume ratio allowing the efficient use of the sample without losses. Additionally, the introduction of the Z-spray ion source on some instruments has enabled an increase in sensitivity. In a Z-spray ion source, the analyte ions follow a Z-shaped trajectory between the inlet tube to the final skimmer which differs from the linear trajectory of a conventional inlet. This allows ions to be diverted from neutral molecules such as solvents and buffers, resulting in enhanced sensitivity. 

Ramsey and Ramseyalso described microchip interfacing to an ion trap mass spectrometer. Microfluidic delivery was realized by electroosmotically induced pressures and electrostatic spray at the channel terminus was achieved by applying a potential between the microchip and a conductor spaced 3-5 mm from the channel terminus. Tetrabutylammonium iodide was tested as a model compound with this device. Later, Ramsey et reported use of a microchip nanoelectrospray tip coupled to a time-of-flight mass spectrometer for subattomole sensitivity detection of peptides and proteins. A fluid delivery rate of 20-30 nL/min was readily obtained by applying an electrospray voltage to the microchip and the nanospray capillary attached at the end of the microfabricated channel without any pressure assistance. 

Wetterhall, M. Nillson, S. Markides, K.E. Bergquist, J. A Conductive Polymeric Material Used for Nanospray Needle and Low-Flow Sheathless Electrospray Ionization Applications, Anal. Chem. 74,239-245 (2002). 

Gatlin, C.L. Kleeman, G.R. Hays, L.G. Link, A.J. Yates, J.R. Protein Identification at the Low Femtomole Level from Silver-Stained Gels Using a New Fritless Electrospray Interface for Liquid Chromatography-Microspray and Nanospray Mass Spectrometry, Anal. Biochem. 263,93-101 (1998). 

---